Characterization of a 1-bp Deletion in the γE-Crystallin Gene Leading to a Nuclear and Zonular Cataract in the Mouse

Abstract

purpose. A previous study had found a mouse mutant to have bilateral nuclear cataract with zonular opacity after paternal irradiation withγ -rays. The mutation was then demonstrated to be allelic with the Cat2 group of dominant cataract mutations and was referred to as Cat2 ^nz in a later study. Because several members of this group have been confirmed as mutations in the gene cluster coding for γ-crystallins (Cryg), these genes were now tested as candidates for Cat2 ^nz .

methods. All six γ-crystallin–encoding genes were amplified by polymerase chain reaction (PCR) from cDNA or genomic DNA and sequenced. An antibody against the changed protein was developed and used for Western blot analysis. The mutant was also characterized morphologically.

results. A 1-bp deletion in exon 2 of the γE-crystallin–encoding gene Cryge was causative of the cataract phenotype. This particular mutation is therefore referred to as Cryge ^nz . The predicted frameshift after codon 29 led to a changed amino acid sequence of 96 amino acids. The altered 13-kDa protein was expressed in the eye lens as demonstrated by Western blot analysis. Cataracts became visible at day 18.5 of embryonic development and reached the final phenotype at 2 weeks after birth.

conclusions. The Cryge ^nz is the sixth mutation in the mouse that has been reported so far to affect the Cryg gene cluster, which demonstrates its importance for lens transparency.

The β- and γ-crystallins were first biochemically characterized as major lens proteins by Mörner ¹ more than 100 years ago. The β- and γ-crystallins belong to a superfamily of proteins that were considered for a long time to be present only in the eye and mainly in the ocular lens. However, just recently, expression of the βB2-crystallin mRNA and protein was reported also in retina, brain, and testis. ²  

The common characteristic of all β- and γ-crystallins is the so-called Greek-key motif. Crystallography has shown that each of theβ - and γ-crystallins is composed of two domains, each built up by two Greek-key motifs. It is widely accepted that β/γ-crystallins evolved in two duplication steps from an ancestral gene coding for a protein folded like a Greek key. The Cryg genes in all mammals consist of three exons: the first one codes for only three amino acids, and the subsequent two are responsible for two Greek-key motifs each. Biochemically, the γ-crystallins are characterized as monomers with a molecular mass of 21 kDa. ^{3 4 5}  

The family of Cryg genes is mainly located in a cluster of six highly related genes (Cryga→Crygf) on mouse chromosome 1 or human chromosome 2q33-35. The seventh Cryg gene (Crygs) is mapped on mouse chromosome 16 or human chromosome 3. Several mutations in the Cryg genes leading to cataracts have been identified. Thus, the mutation ENU-436 affects the Cryga gene, the Nop mutation the Crygb gene, ⁶ and Lop12 the Crygd gene. ⁷ For the Cryge gene, two cataract alleles have been reported so far: the Elo mouse ⁸ and the Cat2 ^t mutant. ⁶ Several hereditary cataracts in humans have been shown to be caused also by mutations in CRYG genes. ^{9 10}  

The mutant Cat2 ^nz was found as a bilateral nuclear cataract with zonular opacity by slit lamp screening of mice after paternal treatment with 2 × 4.55 Gy ¹³⁷Cs γ-ray with a 24-hour fractionation interval. ¹¹ An allelism test of 15 dominant cataract mutations including the nuclear and zonular cataract (Nzc) defined three major cataract groups referred to as Cat2 to Cat4; the Nzc mutation was assigned to the linkage group Cat2 and therefore referred to as Cat2 ^nz . ¹² Because some members of the Cat2 group have been characterized by mutations in the Cryg genes, ⁶ the Cat2 ^nz mutants were tested for an alteration in these genes. Mutational analysis revealed that the second exon of the Cryge gene is affected in this particular mutant line. In addition to this molecular characterization, some morphologic and biochemical characteristics of the mutants are presented. 

Materials and Methods

Animals

The mutation Cat2 ^nz was detected among offspring after paternal radiation exposure in (102/ElxC3H/El)F₁ mice and originally referred to as mutant 116. ¹¹ Cataracts were identified at weaning using a slit lamp (SLM30; Zeiss, Oberkochen, Germany). Homozygous mutant lines have been maintained by brother × sister matings. For control, wild-type C3H/El mice were used. All breedings were performed in the GSF animal facility according to the German Law on the Protection of Animals and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Morphologic Analysis

For gross documentation, lenses were enucleated under a dissecting microscope (MZ APO; Leica, Heidelberg, Germany) and photographed. For detailed histologic analysis, eye globes were fixed for 3 hours in Carnoy’s solution and embedded in JB-4 plastic medium (Polysciences, Eppelheim, Germany) according to the manufacturer’s instructions. Sectioning was performed with a microtome (Ultramicrotome OMU3; Reichert, Walldorf, Germany). Serial transverse 2-μm sections were cut with a dry glass knife and collected in water drops on glass slides. After drying, the sections were stained with methylene blue and basic fuchsin. The sections were evaluated using a light microscope (Axioplan; Zeiss). Images were acquired by means of a scanning camera (Progress 3008; Jenoptik, Jena, Germany) equipped with a screen-capture program (KS100; Carl Zeiss Vision, Hallbergmoos, Germany) and imported into an image-processing program (Photoshop, ver. 5.0; Adobe, Unterschleissheim, Germany). 

Isolation of RNA, DNA, and PCR Conditions

Genomic DNA was prepared from spleen or tail tips of 3-week-old mice according to standard procedures. RNA was isolated from lenses (stored at −80°C) of newborn mice. cDNA synthesis and polymerase chain reaction (PCR) using genomic DNA or cDNA as template were performed essentially as described previously. ⁵ PCR products were sequenced commercially (SequiServe; Vaterstetten, Germany) either after cloning into the pCR2.1 vector (Invitrogen, Leek, The Netherlands) or directly after elution from the agarose gel using kits from Qiagen (Hilden, Germany) or Bio-Rad (Munich, Germany) and subsequent precipitation by ethanol and glycogen. 

Biochemical Analysis of the Lens Extracts

Computer-assisted prediction of biochemical properties of the mutated protein used the Preoteomics tools of the ExPASy Molecular Biology server (Swiss Institute of Bioinformatics, Geneva, Switzerland; available at http://www.expasy.ch). For the hydrophobicity plot, the hypertext for Biomedical Sciences of Colorado State University (Fort Collins, CO) was used (http://arbl.cvmbs.colostate.edu/molkit/hydropathy/index.html). For antibody production against the modified Cat2 ^nz γE-crystallin, a peptide consisting of 15 altered amino acids (Fig. 3) was designed. The peptide synthesis and antibody production were performed commercially (Sigma–Genosys, London, UK). 

For analysis of the lens proteins, 8 to 10 lenses of 3-week-old mice were homogenized in 0.5 ml buffer (50 mM Na₂HPO₄; 100 μM NaCl; pH 7.8). After centrifugation (18,000g), 40 to 60 μg of the supernatant was used for Western blot analysis. Polyacrylamide electrophoresis (12.5% acrylamide), transfer to a nylon membrane (Bio-Rad), and detection of bound antibodies were performed as described previously. ⁶  

General

Chemicals were from Merck (Darmstadt, Germany) or Sigma (Deisenhofen, Germany). The enzymes used for cloning and reverse transcription were from Roche (Mannheim, Germany), and restriction enzymes were from MBI Fermentas (St. Leon-Rot, Germany), if not otherwise mentioned. 

Results

Phenotype and Lens Morphology

Using the slit lamp, the heterozygous Cat2 ^nz mutants could be recognized at eye opening (postnatal day 12). The unique phenotype of a nuclear and a zonular opacity developed in lenses of heterozygous mice (Fig. 1) . Gross morphologic analysis of enucleated eyes from homozygous mice (Fig. 2) demonstrated that the formation of the cataract became visible at embryonic day (E)18.5. At that time, a number of small opaque spots in the center of the lens could be observed. The cataractogenesis progressed to the final phenotype at 2 weeks of age. Both, heterozygotes and homozygous mutants displayed bilateral zonular and nuclear lens opacities. However, the nuclear opacity was more pronounced in homozygous carriers (Fig. 2) . 

Histologic analysis of the Cat2 ^nz lenses in newborn homozygous mutants demonstrated the cataractous core of the lens, which was shifted to the anterior part of the lens (Fig. 3) . In contrast to wild-type lenses, the nuclei of the primary fiber cells were not degraded and were still present in the anterior part of the cataractous region. The lens fiber cells in the central anterior area were swollen. However, the lens bow and the anterior lens epithelium were formed regularly. 

Genomic Analysis

In a previous study, the nuclear and zonular cataract was assigned to the Cat2 group. ¹² We then demonstrated that several members of the Cat2 groups carry a mutation in the Cryg gene cluster coding for γ-crystallins. ⁶ Therefore, we tested also the Cat2 ^nz for a mutation affecting one of the Cryg genes. For this purpose, all six Cryg genes, which have been mapped to mouse chromosome 1, were amplified using gene-specific PCR methods for the cDNA as well as for the genomic DNA. The amplified products were then sequenced. Two polymorphisms, both in exon 3 of the Crygd gene, were observed in our wild-type C3H sequences compared with the sequence data in the database: the first one was observed at positions 310/311 of the database sequence (accession NM 007776) as a change of AG to GA. The corresponding amino acid at codon 100 changed from Val to Met. The second polymorphic site was an A→G at position 495, which led to a replacement of an Arg by an Lys at codon 163. However, both altered amino acids were present at the corresponding positions in the mouse γE- and γF-crystallins. The polymorphism in the exon 2 of the Crygd gene reported by Smith et al. ⁷ was observed in a part of our breeding colonies; however, it remains to be elucidated whether it can be correlated to a particular strain or it is a polymorphism, even within the strains. Nevertheless, it can be concluded that all these polymorphic sites do not have any influence on the function of the γD-crystallin. 

The only difference between wild-type C3H and mutant Cat2 ^nz DNA could be identified in the Cryge gene (accession NM 007777) as a deletion of a T at position 89 of the cDNA (Fig. 4) . The mutation was confirmed by sequencing of exon 2 of genomic DNA from four different animals of the mutant line. It was not found in genomic DNA or cDNA of wild-type animals. Additionally, the mutation destroyed the restriction site for Eco57I and created a new one for DdeI. The loss of the Eco57I site was demonstrated in five homozygous mutants. It was also present in six wild-type mice from different strains (Fig. 5) . Therefore, we conclude that this 1-bp deletion in the Cryge gene is responsible for the cataractous phenotype. The symbol suggested for the new allele is Cryge ^nz . 

Biochemical Analysis

The deduced amino acid sequence demonstrated a frame shift starting in exon 2 after amino acid 29. A new stop codon was present after 96 novel amino acids. Therefore, the mutated protein consisted of 125 amino acids and contained only the first of four Greek-key motifs, which were considered to be important for the regular function of theγ -crystallins. The calculated molecular weight of the novel protein was 13.0 kDa with a predicted isoelectric point at pH 8.4 (wild-typeγ E-crystallin has a theoretical isoelectric point of pH 7.1 and a molecular weight of 21 kDa). Computer-assisted analysis (Proscan) suggested that the novel protein has several phosphorylation sites for casein kinases I and II, protein kinase A, and protein kinase C. 

Analysis for hydrophobicity using the algorithms of Kyte and Doolittle ¹³ revealed two hydrophobic regions, which could be interpreted as membrane-spanning segments. The TMpred program strongly preferred the model of N terminus inside and the first transmembrane domains (amino acids 31–47) from inside to outside. Correspondingly, the second transmembrane region (amino acids 49–73) ran from outside to inside. A similar interpretation was given by the TopPred2 program. The GOR4 secondary prediction program ¹⁴ suggested α-helical regions in the first putative transmembrane domain and in the region between amino acids 97–104. All other regions were thought to be randomly coiled (57%) or extended β-strands (24%). 

Experimentally, we investigated whether the novel protein is expressed in the eye lens and whether it is stable. Therefore, a produced polyclonal antibody against the most hydrophilic region (amino acids 91–105) of the novel protein was used for Western blot analysis of the water-soluble lens proteins. As demonstrated in Figure 6 , the Western blot detected this particular band specifically in the water-soluble lens extract of the mutants, but not in the wild type. Moreover, a degraded product form was visible with a molecular weight estimated at 10 kDa. It is suggested that this smaller band represented a proteolytic digestion product. The size of this fragment is in agreement with the hypothesis that a Lys-C–like endopeptidase activity degrades the novel protein after position 101. The larger fragment obviously can be recognized by the antibody, whereas the smaller fragment containing only four amino acids of the corresponding epitope may not be detected. 

Discussion

We present the molecular and morphologic characterization of a nuclear and zonular cataract mutation in the mouse, which was induced by γ-ray approximately 20 years ago. The γ-rays led to the loss of a single base pair within the Cryge gene. This observation is in line with the current view of the mode of action of ionizing radiation to induce predominantly 1-bp deletions. ¹⁵  

Because the 1-bp deletion changes the open reading frame in exon 2, three of the four Greek-key motifs of the γ-crystallins are not formed. Instead of this, after amino acid 29 a novel protein is formed consisting of 125 amino acids. Western blot analysis demonstrated that the altered protein was stably expressed in the water-soluble compartment of the lens fiber cells. It was obviously partially degraded to a 10-kDa peptide. 

The novel Cryge allele Cryge ^nz leads to a nuclear and zonular cataract in heterozygotes. Only the older lens fiber cells are affected, whereas the outer cortical regions of the lens remain intact. Comparing the histologic observations at this stage, it is very similar to those observed for the Crygb ^nop mutants described previously. ^{6 16 17} The nuclei of the lens fiber cells are not degraded as they usually are at this stage of normal lens development, and they are pushed anteriorly. This implies a disturbance of the terminal differentiation of lens fiber cells. 

However, it is obviously different from other alleles affecting the Cryge gene, such as Cryge ^t or the Cryge ^elo . The recently described cataract mutation Lop12, which was characterized as a mutation in Crygd ⁷ leads to a more severe phenotype, similar to that of the Cryge ^t . ¹⁸  

The Cryge ^nz is the sixth mutation that has been reported to affect the Cryg gene cluster: The Cryge ^{elo 8} and the Crygd ^{Lop12 7} have been reported by other groups; we have characterized the Cryga ^1Neu , Crygb ^nop , Cryge ^t , ⁶ and the Cryge ^ns . ¹⁹ Currently, in humans an increasing number of mutations in the CRYG genes are thought to be associated with cataract formation: the Coppock-like cataract with the CRYGC gene, the aculeiform cataract, and a punctate cataract with CRYGD. ^{9 10} Moreover, a polymorphic congenital cataract was mapped very close to the CRYGB gene. ²⁰ Recently, the list of hereditary human cataracts was extended by the characterization of two further mutations. At first, a 5-bp insertion in the γC-crystallin gene (CRYGC) was reported ²¹ that is associated with a dominant, variable zonular pulverulent cataract. Secondly, a novel CRYGD allele (C109A) leads to an exchange of an Arg residue at position 36 of the γD-crystallin with a Cys and finally to crystallization of the protein in the lens. ²²  

This iterative finding of cataracts associated with mutations affecting one of the Cryg genes makes the CRYG gene cluster to a very interesting locus. At one side, the high number of different clinical phenotypes supports the high importance of these genes for lens transparency. As a common feature so far, they all appear to lead to structural alterations of the proteins. In contrast, the evolutionary knockout of two of these six genes as pseudogenes in man (ψCRYGE, ψCRYGF) suggests that just the loss of function of only a few γ-crystallins may be without consequence. Additionally, there are also several reports both in mouse and human of polymorphic sites within these genes, which could be changed without any effect on the function of the proteins (References ⁵ , ⁶ , and ⁶ and this article). One of the future aspects of γ-crystallin research, therefore, should be a focus on a clear genotype–phenotype correlation of these genes and their encoded proteins. 

 Supported in part by Grant GR 1036/4 from the Deutsche Forschungsgemeinschaft (JG).

 Submitted for publication July 20, 2000; accepted September 6, 2000.

 Commercial relationships policy: N.

 

Figure 1.

 

The nuclear and zonular cataract phenotype. A lens from a 4-week-old heterozygous Cat2 ^nz mutant was dissected and photographed from the anterior surface. The size of the entire lens was 3 mm.

Figure 2.

 

Morphology of cataract formation in the Cat2 ^nz mutant. First signs of cataract formation could be observed in the isolated eyes at E 18.5. Three weeks after birth this process culminated in the presence of a mature cataract. As examples of clear lenses in the wild types, the stages at E 16.5 and E 18.5 are shown. P, postnatal day.

Figure 3.

 

Histology of neonatal lenses. A section through the lens of a newborn mouse is shown, with methylene blue and basic fuchsin staining. The cataractous core of the lens (arrowheads) was shifted to the anterior part of the lens. In contrast to wild-type lenses, the nuclei of the primary fiber cells were not degraded and were present in the lens core (arrows). The lens fiber cells in the central anterior area were disintegrated. (A) Lens from a homozygous Cat2 ^nz mouse (Cryge ^nz ); (B) lens from a wild-type C3H mouse. C, cornea; L, lens; LB, lens bow; LE, lens epithelium; LFC, lens fiber cells; P, postnatal day.

Figure 4.

 

Sequence analysis of the Cat2 ^nz mutant. The Cryge DNA sequence from the wild-type mouse (top sequence) compared with that of the Cat2 ^nz mutant (bottom sequence). The deduced amino acid composition is demonstrated above or below the DNA sequence. The place of the mutation within exon 2 is indicated. The deletion of the T at position 89 led to a change in the open reading frame. The mutated DNA sequence encoded a novel peptide consisting of 125 amino acids (in bold). The peptide used for antibody production is underlined. The Eco57I recognition site (gray box) was destroyed by the mutation. Arrow: cutting site.

Figure 5.

 

Cryge digest by Eco57I. The exons 1 and 2 as well as the connecting intron A were amplified from genomic DNA. The PCR fragment was analyzed by agarose electrophoreses with (+) and without (−) subsequent digestion by Eco57I. The genomic DNA from all wild types (top) could be digested, but not the DNA from the homozygous Cryge ^nz mutants (bottom; number above the lanes indicates the subject). According to the information from the supplier, the cleavage of DNA by Eco57I is never complete. M, marker; PCR, PCR product; RE, PCR product after cleavage by Eco57I.

Figure 6.

 

Western blot (left) of lens extracts. Extracts of water-soluble lens proteins (40 and 60 μg) were separated by polyacrylamide gel electrophoresis (right) and analyzed for the presence or absence of the altered γE-crystallin using a specific antibody. The novel protein could be detected as a 13-kDa band (arrow) solely in the cataractous lenses and not in the lenses from the C3H wild type. Because the 13-kDa band is in agreement with the calculated molecular mass of the entire protein, the second band at 10 kDa (arrowhead) may represent the N-terminal fragment after cleavage by lenticular Lys-C endopeptidase activity.

The authors thank Erika Bürkle, Dagmar Reinl, and Monika Stadler for expert technical assistance. Oligonucleotides were obtained from Utz Linzner, GSF-Bioinformatics Group, AG BIODV.