Crystallin (cid:1) B-I4F Mutant Protein Binds to (cid:2) -Crystallin and Affects Lens Transparency*

A new mouse mutant line , Clapper , identified from N -ethyl- N -nitrosurea (ENU)-mutagenized mice, develops a dominant lamellar cataract. The cataract blocks the image of retinal fundus and transmits a fuzzy fluorescein image of retinal vasculature during angiography. The cataractous lens opacity decreases as the mice age. The C lapper mutation has been identified to be a missense mutation of the (cid:1) B-crystallin gene that replaces the 4th isoleucine residue with a phenylalanine ( (cid:1) B-I4F). Unlike wild type (cid:1) B, the (cid:1) B-I4F mutant protein binds to (cid:2) -crystallin to form high molecular weight complexes in vivo and in vitro . Circular dichroism measurements indicate that (cid:1) B-I4F protein is less stable than wild type (cid:1) B at high temperature. Darkly stained aggregates, enlarged interfiber spaces, and disorganized residue in members of the Cryg family, is substituted with a phenylalanine (Fig. 2 B ). No additional mutation was detected in the other five members ( (cid:3) A, (cid:3) C, (cid:3) D, (cid:3) E, and (cid:3) F) in the cluster (data not shown). (cid:3) B-I4F Mutant Subunits Bind to (cid:1) -Crystallin to Form High Molecular Weight Complexes— Gel filtration analysis shows an obvious reduction of both the (cid:1) -crystallin peak and the (cid:3) -crys-tallin peak in the lens water-soluble protein fraction of heterozygous or homozygous mutant mice when compared with that of wild type mice (Fig. 3 A , a ). The changes of the (cid:1) -crys-tallin peak are similar for heterozygous and homozygous lenses, whereas the (cid:3) -crystallin peak is obviously more reduced in the homozygous mutant compared with the heterozygous mutant. The (cid:2) -crystallin peak seems unchanged in all the samples. The eluted (cid:1) -crystallin peaks were collected and further analyzed by Coomassie-stained SDS-PAGE gels, Western blotting, and 2-DE. A representative Coomassie Blue-stained gel shows that the (cid:1) -crystallin peak of heterozygous mutant lens contained normal (cid:1) A-, (cid:1) A INS -, and (cid:1) B-crystallins, plus additional protein bands that could be recognized by an anti- (cid:3) -crystallin polyclonal antibody (Fig. 3 A , b and c ). Similar results were obtained from the homozygous lens samples (data not Thus, the altered (cid:1) -crystallin peak contains (cid:3) -crystallin.

Transparency and a high refractive index are essential features for the function of the lens (1). Age-related cataracts, clinically named for lens opacities in elderly people, are one of the major causes for vision impairment in the world. Abnormal protein modifications and/or morphological changes in the lenses are associated with this disease, but the causative mechanism is not well understood.
The vertebrate lens consists of bulk elongated fibers covered with a monolayer of epithelial cells on the surface of the anterior hemisphere. A thick basement membrane called the lens capsule, mainly consisting of collagens, wraps both the lens epithelium and fibers. The center core of the lens consists of primary fibers differentiated from the posterior cells of the lens vesicle at an early embryonic stage. The rest of the lens fibers are secondary fibers differentiated from equatorial epithelial cells. The newly formed secondary fibers lay on the top of the previous generation of fibers in a concentric manner at the peripheral equatorial region of the lens. These fibers eventually eliminate their intracellular organelles to become mature fibers in the inner region of the lens (2).
Lens proteins are mainly composed of three groups of crystallins, ␣, ␤, and ␥ (1). The ␣-crystallins form high molecular complexes (ϳ300 -1000 kDa), consisting of three isoforms, ␣A, ␣A ins , and ␣B, encoded by the CryaA and CryaB gene, respectively. They are members of the small heat shock protein family and act as protein chaperones to prevent the aggregation of misfolded proteins in the lens (3,4). Genetic studies show that mutations in either CryaA or CryaB cause dominant or recessive cataracts in humans and mice. In CryaA, two missense mutations (␣A-R49C and ␣A-R116C) in humans and one missense mutation (␣A-V124E) in mice result in dominant nuclear cataracts (5)(6)(7), while one nonsense mutation (␣A-W9X) in humans and a missense (␣A-R54H) and a null mutation (knock-out) in mice result in recessive cataracts (8 -10). In CryaB, a frameshift mutation and a missense mutation (␣B-R120G) result in dominant cataracts in humans (11,12), but ␣B (Ϫ/Ϫ) knock-out mice develop normal lenses (13). Therefore, ␣A-crystallin is essential for lens transparency, whereas the role of ␣B-crystallin is not clear. Based on in vitro biochemical assays, both ␣A and ␣B mutations cause either a decrease of chaperone activity of the protein or an increase of protein insolubility and/or instability.
Both ␤and ␥-crystallins are lens structural proteins and belong to the members of a related ␤/␥-superfamily that shares a common structural character: a Greek key domain (1). These crystallins are most likely evolved from a duplication of an ancestral protein folded like a Greek key. Six members of the ␤-crystallin family are divided into two subgroups, the more acidic (␤A-) and the more basic (␤B-) proteins. Each subgroup is encoded by three genes: CrybA1, CrybA2, and CrybA3, or CrybB1, CyrbB2, and CrybB3. Also, CrybA1 encodes two proteins, ␤A1 and ␤A3. Mutations of CrybA1/A3, CrybB1, and CrybB2 have been reported to cause dominant cataracts in humans and mice (14 -21). However, it is not known whether ␤and ␥-crystallins play additional functions besides acting as necessary structural components for lens transparency.
Many mutations of different ␥-crystallins have been reported to cause various types of cataracts in humans and mice (22)(23)(24). The ␥-crystallin family has seven members, ␥A, ␥B, ␥C, ␥D, ␥E, ␥F, and ␥S, encoded by its own gene (CrygA-F and CrygS). CryA-F genes are located in a cluster at 32 cm on mouse chromosome 1 or at the long arm region (q33-35) on human chromosome 2. CrygS is located on mouse chromosome 16 or on human chromosome 3. Missense mutations of CrygC and CrygD and an activation of pseudo-CrygE gene cause dominant cataracts in humans (25). At least 22 mutations of different Cryg isoforms have been reported to cause a broad range of lens phenotypes in mice (24).
In vitro biochemical and structural studies of different ␥-crystallin mutants have verified that the mutations can cause the conformation changes of their protein structure and initiate abnormal protein-protein interactions to form waterinsoluble aggregates (26), and these aberrant protein aggregates could cause the light scattering in the lens. However, in vivo results show that different ␥-crystallin mutations cause these unique lens phenotypes via distinct mechanisms. One report showed that the crystallization of ␥D-R36S mutant protein is the mechanism for its unique cataract formation (27,28). In a mouse ␥B mutation, a substitution of the C-terminal 27 amino acid residues by 6 new amino acid residues in ␥Bcrystallin caused the formation of large inclusions in the nuclei of lens fiber cells (29). This ␥B mutation was hypothesized to disrupt nuclear function, which causes lens phenotypes. Therefore, we hypothesize that some of the ␥-crystallin mutant proteins cause cataracts by specifically perturbing their unknown in vivo functions that are essential for lens development or transparency.
Here we report a dominant lamellar cataractous mouse line that was identified from a screen for lens phenotypes of ENUinduced mouse germ line mutations. The causative gene is a new point mutation of the CrygB gene with the 4th isoleucine substituted by a phenylalanine. This mutation presents a novel model to study a unique lamellar cataract that decreases with age. We have investigated the molecular basis for how ␥B-I4F mutation specifically leads to its distinctive lens phenotype.

ENU-induced Mouse Mutations, Genomic Linkage Analysis, and
Causative Gene Identification-The ENU-mutagenized mice were produced as reported previously (30). Mouse breeding and genomic linkage analysis of this study were performed using an identical approach described previously (31). The dominant mutation Clapper, mouse line L10 at C57BL/6J (B6) strain background, was crossed with wild type C3H/HeN mice to produce affected G1 hybrid mice that were further crossed with wild type C3H/HeN animal again to produce secondary generation (G2) mice. The G2 mice were phenotyped and tailed for genomic DNA that was used for a genome-wide linkage analysis by using a total of 59 microsatellite markers (31). Based on the chromosomal location, causative gene candidates were identified from the mouse genome data base at the National Center for Biotechnology Information web site. DNA sequencing was performed on DNA products generated from the transcripts of causative gene candidates by reverse transcription PCR and/or from the exons of mutant genomic DNA by PCR. The primers used for DNA sequencing analysis of different ␥-crystallin genes are listed in Table I. Two sets of primers were used to sequence the exons of the ␥B-crystallin gene of the genomic DNA samples isolated from the tails of the Clapper homozygous mutant and wild type mice at B6 background, respectively.
Cloning and Expression of Recombinant Wild Type and Mutant ␥B-Crystallin-Two sets of the DNA primers, one for wild type cDNA (CryGbhindIII-left and CryGb-right) and one for mutant cDNA (CryGbbamhl-left and CryGb-right), were designed to clone wild type and mutant ␥B-crystallin genes, respectively ( Table I). The 5Ј-end primer for wild type ␥B gene contained the translational initiation site ATG with a HindIII site for cloning, whereas the 5Ј-end primer for ␥B mutant contained the mutated nucleotide (T) for encoding phenylalanine and a BamHI site for cloning. A common 3Ј-end primer was used in both sets of reverse transcription PCR reactions. A kit (first-strand synthesis system for reverse transcription PCR, catalogue number 11904 -018; Invitrogen) was used to synthesize the first-strand cDNA from total RNA isolated from the lenses of B6 mice, and then the Platinum Pfx DNA polymerase kit (catalogue number 11708 -039; Invitrogen) was used to amplify the full-length of cDNA, 605-bp fragments, of wild type and mutant ␥B. Both cDNA fragments were subcloned into the PCR-Blunt II-TOPO vector from the Zero Blunt TOPO PCR cloning kit (catalogue number K2800 -20; Invitrogen).
Phenotypic Screening, Fundus Pictures, and Fluorescein Angiography-Mouse pupils were dilated by using 1% phenylephrine with 1% atropine before mice were visually examined for the clarity of the lens through a slit lamp and for the retinal fundus by using an indirect ophthalmoscope. The cataract was directly imaged from living animals by a slit lamp (Nikon-FS3) with a camera. Fresh lenses, dissected from enucleated eyeballs of wild type and mutant mice, were imaged under a Leica MZ16 dissecting scope using a digital camera.
Fluorescein angiography protocol was as follows. Fundus pictures of mouse retina were taken by a Genesis small animal fundus camera (Kuwa) that was specially designed for mice (32). Fluorescein angiography was taken by the same instrument with one specifically manufactured power pack linked with a camera containing a barrier filter. Mice were intraperitoneally injected with sodium fluorescein at a dose of 0.01 ml (25% pharmaceutical grade sodium fluorescein; Akorn Inc.)/5 g body weight. The retinal vasculature was filled with dye in less than a minute after sodium fluorescein administration, and the photos were taken by using Kodak elite2 200 ASA color slide films for fundus images and 800 ASA for angiography. A timer on the power pack was used for recording elapsed time, in seconds, on each photo.
SDS Gel Electrophoresis, Coomassie Blue Staining, Western Blotting-Analysis of lens total protein was as follows. Fresh lenses were dissected from enucleated eyeballs and quickly weighed for their wet weight. Lenses were then homogenized based on the ratio of 40 mg lens wet weight/ml of sample buffer (60 mM Tris-HCl, pH 6.8, 2% SDS, 10 mM dithiothreitol, 10% glycerol, and 0.0001% bromphenol blue). Homogenates were sonicated before loading into the gels. Analysis of lens water-insoluble proteins was as follows. One fresh lens was homogenized in 0.5 ml of 0.1 M NaCl and 50 mM Na 2 HPO 4 (pH 7.2) buffer, and then the lens homogenate was centrifugalized to collect the insoluble pellet at 15,000 rpm for 15 min. The pellet was washed with the same buffer twice and then dissolved in 50 l of sample buffer. Equal volume of the samples was loaded into a 12.5% SDS-PAGE gel for separation, and then proteins were detected by Coomassie Blue staining or Western blotting. At least three independent experiments of lenses from different mice were carried out for the quantification of these biochemical assays.
Expression and Purification of Recombinant Wild Type and Mutant ␥B (I4F)-Wild type ␥B and mutant ␥B-I4F cDNAs were subcloned into a bacterial expression plasmid and prepared as described previously (33). Recombinant wild type ␥B-crystallin were expressed as soluble proteins, but ␥B-I4F mutant proteins were exclusively in the inclusion bodies as insoluble proteins. Small amounts of soluble ␥B mutant proteins were obtained by solubilizing the inclusion bodies in 8 M urea followed by stepwise dialysis against buffer as described before (34). The ␣-crystallin was prepared from bovine lenses and purified as described (35).
Gel Filtration Chromatography, Two-dimensional Electrophoresis , and Circular Dichroism (CD)-An Amersham Biosciences Superose 6 HR 10/30 column connected to the AKTA FPLC system was used for gel filtration analysis of lens water-soluble homogenates and the mixtures of bovine ␣-crystallin and recombinant ␥B-crystallin. 2-DE 1 was performed based on a protocol provided by Bio-Rad using PROTE-AN-IEF Cell with 11-cm IPG strips, pH 3-10 (Bio-Rad), and 8 -16% linear gradient precise gels were used for the second dimensional analysis. The gels were stained with Coomassie Blue. CD studies were performed by using a JASCO model J-810 Spectropolarimeter with the Peltier temperature control system (JASCO-PTC-348WI).
Morphological and Immunohistochemical Analysis-Standard histology was as follows. Enucleated eyeballs were fixed in Canoy's fixative solution, a mixture of ethanol and acetic acid (3:1), for overnight before processing using a standard histological method for paraffin-embedded samples. The sections were stained with hematoxylin and eosin. Thin sectional analysis under light and electron microscopy was as follows. Anterior chambers of enucleated eyeballs of 3-week-old mice were opened immediately after enucleation and then immersed in a fixative solution containing 2% glutaraldehyde and 2.5% formaldehyde in 0.1 M cacodylate buffer (pH 7.2) at 30°C for 2 days and thereafter at room temperature for an additional 3 days. This fixative procedure was modified from a method described previously to prevent protein aggregation caused by "cold" cataract (36). Eye cups were postfixed in 1% aqueous OsO4, stained en bloc with 2% aqueous uranyl acetate, and then dehydrated through graded acetone. Samples were embedded in eponate 12-araldyte 502 resin (Ted Pella, Redding, CA). Thin sections (1 m) across the equatorial plane were collected on glass slides and stained with Toluidine blue. Bright field images were acquired via a Zeiss Axiovert 200 light microscope with a digital camera. Ultrathin sections (60 nm) were collected and stained with Sato's triple lead solution before being examined under a JEOL JEM-1200EX II electron microscope (JEOL, Tokyo, Japan). Immunohistochemical staining was as follows. A previously described method was used for the immunohistological analysis (37). Fluorescent images were collected under a Zeiss Axiovert 200 fluorescent microscope with an Axiocam camera.

Clapper Mutation Develops a Unique Lamellar Cataract-A
female founder of the Clapper mutation (also called mouse line L10) was identified from ENU-induced F1 mutant mice in the C57BL/6J (B6) strain by slit lamp examination. This mutation develops cataracts similar to human hereditary lamellar cataracts (Fig. 1A). The cataracts can be observed in both heterozygous and homozygous mutant mice at weaning age. The homozygous lenses show a much denser opacity than the heterozygous lenses (Fig. 1B). The cataract in 3-week-old ho-mozygous mutant mice occupies the inner region and covers ϳ75% of the lens, reaching about 675 Ϯ 5 m radius distance from the lens center where the lens radius is ϳ900 m. The lens periphery, ϳ25% of the lens and ϳ225 Ϯ 5 m radius distance from the lens capsule, remains clear.
Because of the nuclear cataract (Fig. 1E2), the retinal vasculature of the homozygous mutant mice at age 3 weeks cannot be seen through the fundus imaging (Fig. 1C2), but the fluorescein angiography shows a fuzzy image of its retinal vasculature (Fig. 1D2). We have also found that this cataract decreases in opacity as homozygous Clapper mice grow old (Fig.  1E3) and that retinal vessels could be seen in the fundus examination (Fig. 1C3). Moreover, a focused angiography was obtained from old homozygous Clapper mice (Fig. 1D3). The images of retina fundus, angiography, and the lens of a wild type mouse are shown as the controls (Fig. 1, C1, D1, and E1).
CrygB-I4F Is the Causative Gene Mutation-Using 48 meioses from a backcross between heterozygous Clapper (L10) mice at C57BL/6J strain background and wild type C3H/HeJ mice, we have mapped the Clapper mutation to chromosome 1 near the linkage marker D1Mit380 with a Lod score of 4.6 by a genome-wide linage analysis ( Fig. 2A). After searching the mouse genome data base at the National Center for Biotechnology Information, we found crystallin Cryg gene cluster encodes six ␥-crystallin isoforms, ␥A, ␥B, ␥C, ␥D, ␥E, and ␥F, in close proximity to this marker. Previous studies have shown that mutations in different Cryg members cause cataracts in humans and mice. Therefore, a DNA sequencing analysis was performed to verify the coding regions of these six ␥-crystallin genes by using either reverse transcription PCR of their lens transcripts or PCR of their exons from the genomic DNA of homozygous mutant mice. We have found a missense mutation (A 3 T) in the CrygB gene. This point mutation results in a mutated ␥B-crystallin in which the 4th isoleucine, a conserved 1 The abbreviation used is: 2-DE, two-dimensional electrophoresis. residue in members of the Cryg family, is substituted with a phenylalanine (Fig. 2B). No additional mutation was detected in the other five members (␥A, ␥C, ␥D, ␥E, and ␥F) in the cluster (data not shown).

␥B-I4F Mutant Subunits Bind to ␣-Crystallin to Form High Molecular Weight
Complexes-Gel filtration analysis shows an obvious reduction of both the ␣-crystallin peak and the ␥-crystallin peak in the lens water-soluble protein fraction of heterozygous or homozygous mutant mice when compared with that of wild type mice (Fig. 3A, a). The changes of the ␣-crystallin peak are similar for heterozygous and homozygous lenses, whereas the ␥-crystallin peak is obviously more reduced in the homozygous mutant compared with the heterozygous mutant. The ␤-crystallin peak seems unchanged in all the samples. The eluted ␣-crystallin peaks were collected and further analyzed by Coomassie-stained SDS-PAGE gels, Western blotting, and 2-DE. A representative Coomassie Blue-stained gel shows that the ␣-crystallin peak of heterozygous mutant lens contained normal ␣A-, ␣A INS -, and ␣B-crystallins, plus additional protein bands that could be recognized by an anti-␥-crystallin polyclonal antibody (Fig. 3A, b and c). Similar results were obtained from the homozygous lens samples (data not shown). Thus, the altered ␣-crystallin peak contains ␥-crystallin.
Compared with the 2-DE results of wild type controls (Fig.  3B, a and b), an additional protein spot shows in the 2-DE data of the eluted ␣-crystallin peak and the total lens-soluble protein fraction of the heterozygous Clapper mutant (Fig. 3B, c  and d, indicated by an arrow). Thus, we carried out a protein identification of this additional protein spot from 2-DE gels and the additional bands from the Coomassie Blue-stained onedimensional gel by using mass spectrometry-mass spectrometry analysis. The results of both protein identification analyses verified that this additional protein was ␥B-crystallin (data not shown). These data suggest that ␥B-I4F mutant proteins bind to ␣-crystallins to form high molecular weight water-soluble complexes in the ␣-crystallin peak.
To understand why ␥B-I4F binds to ␣-crystallin in vivo, we carried out an in vitro study for the interaction between bovine ␣-crystallin and recombinant ␥B-I4F mutant with recombinant wild type ␥B as control. The bovine ␣-crystallin was purified from bovine lenses, and the recombinant ␥-crystallin proteins were purified from a bacteria expression system. At 37°C, neither wild type ␥B nor mutant ␥B-I4F bound to ␣-crystallin as shown by the gel filtration analysis after they mixed with ␣-crystallin separately (data not shown). Because the ␥-crystallin is a relatively heat-stable protein, we decided to slightly increase the temperature from 37 to 45°C to see if the mutant protein behaves like the wild type protein. The results showed that the ␥B-I4F mutant protein binds to ␣-crystallin, but the wild type ␥B does not bind to ␣-crystallin at 45°C (Fig. 3A, d). This in vitro result confirms the in vivo data that the ␥B-I4F mutant protein binds to ␣-crystallin to form large aggregates.
␥B-I4F Mutant Protein Is Less Heat-stable and an Increase of Water-insoluble Proteins Leads to Cataract Formation-CD measurements were carried out to examine the structure and stability of the recombinant ␥B-I4F mutant protein. The far-UV CD spectrum of ␥B-I4F mutant was identical to that of wild type ␥B (data not shown). This indicates that the secondary structure of the ␥B-I4F mutant was similar to that of wild type. The near-UV spectra reflect the tertiary structure mostly contributed by aromatic amino acid residues. The wild type ␥B protein is known to be stable (1). This is evident by the unchanged near-UV CD spectra between 25 and 50°C (Fig. 4A). The near-UV CD of the ␥B-I4F mutant were essentially the same as that of wild type ␥B at 25 and 40°C; however, at 50°C, the ␥B-I4F mutant completely lost its tertiary structure as evidenced by the near-UV CD spectrum (Fig. 4B). The ␥B-I4F mutant protein begins to aggregate and precipitate at 45°C (data not shown). These data indicate that the ␥B-I4F mutant is significantly less stable than wild type ␥B.
Lens protein analysis shows an increase of water-insoluble proteins (molecular mass between 20 and 30 kDa) in heterozygous and homozygous lenses (Fig. 3B, e). The homozygous lenses contain more insoluble proteins than the heterozygous lenses. Western blotting results further show an obvious increase of different crystallin proteins, including ␣A-, ␣B-, and especially ␥-, crystallins in the insoluble fraction (Fig. 3B, f). In addition, we have found a 15% reduction of the ␣8 (Cx50) connexin in the homozygous lens homogenates, but no detectable change of ␣3 (Cx46) connexin and MP26 (aquaporin 0), when compared with that of wild type and heterozygous lenses (Fig. 3B, f). The quantitative results were based on the average densitometric measurements of three independent Western blotting results of these lens-insoluble fractions prepared from three different sets of lenses.
Clapper Lenses Show Relatively Normal Histology and Connexin Distribution-Representative histological sections with hematoxylin and eosin staining show similar cellular morphology in the bow regions of wild type, heterozygous, and homozygous mutant lenses (Fig. 5A). Immunohistochemical staining of ␣3 and ␣8 connexins demonstrated that the punctate fluorescent signals of ␣3 and ␣8 connexins in heterozygous or homozygous lens sections were similar to that of wild type lens sections (Fig. 5B). However, we consistently observed that the distribution of ␣8 staining signals appears to be less uniform in homozygous lens sections. This minor change of ␣8 staining is correlated to the previous Western blotting result that shows an ϳ15% reduction of ␣8 protein in the lens (Fig. 3B, f).
Enlarged Interfiber Spaces and Aggregates Present in the Clapper Lenses-We have further examined the morphological differences among wild type, heterozygous, and homozygous lenses by using a plastic-embedded and thin sectional method. Peripheral cortical fibers appear normal in heterozygous and homozygous lenses through an equatorial cross-section (Fig.  6A). However, under the light microscope, dark-stained aggregates are observed in the deep cortex of the homozygous lens, especially in the region of 180 -280 m from the lens capsule. These aggregates are not often seen in the heterozygous lens. Disorganized, irregularly shaped, and smaller fibers are also observed in the deeper regions of both heterozygous and homozygous lenses (ϳ380 -480 m from the capsule in a crosssection). The inner fibers of homozygous lenses show more severe changes than that of the heterozygous lenses.
Under the electron microscope, we observed fiber-to-fiber junctions with almost no interfiber spaces between lens fiber cells in the peripheral cortex, up to 120 m from the lens capsule, of both heterozygous and homozygous lenses (Fig.   6B). However, interfiber spaces became visible at the region ϳ180 m from the capsule, especially in the homozygous mutant lens. Obvious enlarged interfiber spaces appear at the region ϳ330 m from the capsule (Fig. 6B). These enlarged interfiber spaces were directly measured from the photos of the thin sections for a statistical analysis. The average width of the interfiber spaces, located 330 -350 m from the lens capsule in both heterozygous and homozygous lenses, is ϳ40 -50 nm. That is ϳ3 times wider than that of wild type lenses with 15 nm in width at the same location (Fig. 6C).   FIG. 3. A, the interaction between ␥B-I4F and ␣-crystallin. a, gel filtration graphs show a reduction of ␣and ␥-crystallin in lens water-soluble fractions of both heterozygous (I4F/ϩ) and homozygous (I4F/I4F) mice when compared with that of wild type (ϩ/ϩ) mice at the age of 3 weeks. b, the ␣-crystallin peaks were collected and examined by SDS gel with Coomassie Blue staining. Results show additional bands (indicated by arrowheads) in the ␣-crystallin peak of heterozygous (I4F/ϩ) lenses compared with wild type (ϩ/ϩ) control, which consists of three ␣-crystallin subunits, ␣A (bottom band), ␣B (middle band), and ␣A ins (upper band). c, Western blotting shows these additional bands in panel B are recognized by a rabbit polyclonal anti-␥-crystallin antibody. The ␣B-crystallin, recognized by anti-␣B antibody, is shown as a reference. d, gel filtration chromatograms show that the ␣-crystallin peak becomes larger with high molecular weight components in the mixture of native bovine ␣-crystallin with recombinant ␥B-I4F mutant proteins, but not in the mixture with recombinant wild type ␥B. The mixtures of 0.4 mg of bovine ␣-crystallin and 0.4 mg of recombinant (wild type or mutant) ␥B-crystallin at the ratio of 1:1 at the 1.6 mg/ml concentration were treated at 45°C for 30 min before being loaded into the size filtration column. B, biochemical changes of the Clapper lenses. a-d, 2-DE results of the ␣-crystallin peak (panel a, 1, ␣A-crystallin; 2, ␣A ins ; 3, ␣B-crystallin; 10 g of proteins loaded) collected after a gel filtration analysis of the lens water-soluble proteins (panel b, 15 g of proteins loaded) of 3-week-old wild type mice and of ␣-crystallin peak (panel c, 10 g of proteins loaded) collected after a gel filtration analysis of the lens water-soluble proteins (panel d, 15 g of proteins loaded) of 3-week-old homozygous Clapper mice. Arrows indicate new protein spots. The pH gradient is from 3 on the left side to 10 on the right side of the 2-DE images. e, a Coomassie Blue-stained gel shows the water-insoluble proteins of wild type (ϩ/ϩ), heterozygous (I4F/ϩ), and homozygous (I4F/I4F) lenses of mice at 3 weeks of age. Mutant samples show an increase of several proteins located between molecular mass markers 21 and 29 kDa. The total insoluble pellet of one lens was dissolved in 50 l of 1ϫ loading buffer. 20 l of each sample was loaded onto 12.5% PAGE gel for separation and analyzed by Coomassie Blue staining. f, the same samples as in panel e were diluted 10-fold, and then 20 l/sample was loaded on a gel for Western blotting with antibodies against ␣A-, ␣B-, and ␥-crystallin. 20 l of total lens homogenates/sample, with the ratio of 40 mg lens wet weight/ml of sample buffer, were loaded for detecting ␣3-, ␣8-connexin, and MP26 (aquaporin 0) by Western blotting.

DISCUSSION
␥-Crystallins are one class of major structural proteins responsible for the lens transparency. The differential expression of six different ␥-crystallin isoforms (␥A-␥F) during lens development suggests that ␥-crystallin diversity might be important for establishing and maintaining the lens transparency (38,39). However, it is not known how ␥-crystallin isoforms assemble with each other or with other lens proteins to form shortrange ordered structures in lens fibers. Based on the sequence similarities among different ␥-crystallin isoforms, a common tertiary structure is predicted, with most of the hydrophobic residues buried in the center of the three-dimensional structure and the charged residues located mainly on the surface (1). The unconserved amino acids among ␥-crystallins may provide diversity in surface properties to allow their unique intermolecular contacts with other proteins in the lens. Therefore, the combination of high stability and solubility of ␥-crystallins is necessary for lens transparency.
In this study, we identified a new cataractous mutation, Clapper, caused by a point mutation of the ␥B-crystallin gene that resulted in the 4th amino acid residue isoleucine (I) being substituted by phenylalanine (F). This ␥B-I4F mutation led to a unique lamellar cataract. At present, we do not have a reliable method to quantitate the changes of lens opacity in mouse in vivo. Thus we used both fundus and angiographic images to directly evaluate the qualitative changes in lens cataracts. It is unexpected that the lens opacity decreases as the mice age. A relatively focused image of retinal angiography was also obtained from the old mice. Therefore, Clapper mutation provides a unique cataract model that differs from all previous crystallin mutations, including ␥B nop mutation, which caused severe growth defects (40), as well as the Opj cataract caused by the ␥S-F9S mutation (41).
The 4th amino acid residue isoleucine (I), a conserved residue in all six ␥-crystallins isoforms (␥A-␥F), is located in the first ␤-strand of the Greek key motif. This residue is completely buried in the hydrophobic environment (42). The substituted phenylalanine did not affect the secondary structure and the tertiary structure of ␥B at 25 and 40°C as evidenced by CD spectra. However, recombinant ␥B-I4F mutant protein is unstable at 45°C, completely loses its tertiary structure at 50°C, and mainly forms inclusion bodies as insoluble proteins in the bacterial expression system. Thus, the recombinant ␥B-I4F mutant protein behaves differently from the previous mutations, including ␥S-F9S, ␥D-P23T, and ␥C-T5P. The ␥S-F9S mutant proteins are unstable but soluble in the bacterial expression system (41). The human ␥D-P23T is stable but less soluble (43). The ␥C-T5P mutation destabilizes the protein by a conformational change (44). Recombinant ␥B-I4F does not bind to ␣-crystallin at 37°C in vitro, whereas the native ␥B-I4F mutant binds to ␣-crystallin to form high molecular weight complexes in vivo. One of the explanations for this discrepancy is that ␣-crystallins do recognize the very subtle changes of ␥B-I4F in vivo at 37°C because this protein is exposed to this temperature for days and weeks. ␣-Crystallin has been hypothesized to be "a molecular sensor" that can bind proteins with very subtle conformational changes. These were demonstrated in previous studies of ␤B2 and T4 lysozyme mutations (45,46). The present study supports this hypothesis.
This nuclear cataract is associated with a loss of watersoluble ␥-crystallin, an increase of water-insoluble crystallins, and especially the appearance of the enlarged interfiber spaces between lens inner mature fibers. Thus, this cataract is, at least partly, caused by the light scattering of the protein aggregates consisting of ␥B-I4F proteins and ␣-crystallins. However, it is not understood why the nuclear cataract reduces its opacity as Clapper mice grow old, because abnormal protein aggregates usually increase lens opacity with age.
It is also not known how ␥B-I4F mutation leads to enlarged interfiber spaces in inner fibers or whether this alteration is because of the indirect consequence of a functional loss of ␣-crystallin due to binding with ␥B-I4F mutant proteins. Normal mice are born with opaque lenses, and their lenses become clear in 2 weeks after birth. The narrowing of the interfiber spaces is one of the cellular processes to ensure lens transparency during development. The water channels consisting of aquaporin 0 (MP26 or MIP) and intercellular gap junction channels consisting of connexins in the plasma membranes of lens fibers have been implicated as the main structural components that mediate the narrowing of the interfiber spaces between mature fibers in the lens (2,47,48). Although in our study a slight reduction of ␣8 connexin was detected in the homozygous lens, there is no evidence that shows interactions between connexins and ␥or ␣-crystallins. A recent study reported that the C-terminus of aquaporin 0 (MP26) directly interacts with ␥E-crystallin to recruit ␥E to the plasma membrane. ␣-Crystallins also have a functional role in preserving the integrity of the plasma membrane (49). We hypothesize that the unusual interaction between ␥B-I4F and ␣-crystallins results in the changes of plasma membrane of mature fiber cells. It might be important to determine the ultrastructural arrangements of water channels (MP26) and gap junctions in the plasma membrane of the mature fibers of Clapper lenses by using freeze-fracture electron microscopy analysis in the future.
At present, we do not know the mechanisms for why mutant lens quality improves with age. We believe it is important to further study the molecular basis for the interaction between ␥B-I4F and ␣-crystallin and to investigate the molecular and cellular mechanisms that are responsible for the decrease in lens opacity with age.
FIG. 6. Light and electron micrographs show unique morphological changes in mutant lenses. A, light micrographs of the lens plastic sections show the fiber organization of 3-week-old wild type (ϩ/ϩ), heterozygous (I4F/ϩ), and homozygous (I4F/I4F) lens cross-sections around the lens equator, with the distances from lens capsule (50 -150, 180 -280, and 380 -480 m, respectively). Some dark-stained droplets (indicated by arrowheads) appear only in the restricted inner region (180 -280 m) of the homozygous lens section. Scale bars, 50 m for the left panels and 10 m for all other panels. B, electron micrographs show the interfiber spaces between the lens fibers of wild type, heterozygous, and homozygous lenses with the distances from the equatorial capsule (60, 90, 120, 180, 330 m). Enlarged interfiber spaces can be seen in the region ϳ180 -330 m from the lens capsule of heterozygous and homozygous lenses. C, the interfiber spaces were measured from the electron microscope micrographs in the areas 330 -350 m away from the lens capsule. Statistical results show that the interfiber space in both heterozygous and homozygous lenses is ϳ3 times wider than that in wild type control (p Ͻ Ͻ-0.001). Scale bar, 200 nm.