Characterization of a New, Dominant V124E Mutation in the Mouse αA-Crystallin–Encoding Gene

Abstract

purpose. During an ethylnitrosourea (ENU) mutagenesis screening, mice were tested for the occurrence of dominant cataracts. The purpose of the study was morphologic description, mapping of the mutant gene, and characterization of the underlying molecular lesion in a particular mutant, Aey7.

methods. Isolated lenses were photographed and histologic sections of the eye were analyzed according to standard procedures. Linkage analysis was performed with a set of microsatellite markers covering all autosomal chromosomes. cDNA was amplified after reverse transcription of lens mRNA. For PCR, cDNA or genomic DNA was used as a template.

results. Nuclear opacity and posterior suture anomaly were visible at eye opening and progressed to a nuclear and zonular cataract at 2 months of age. The opacity as well as the microphthalmia was more pronounced in the homozygotes than in the heterozygotes. The mutation was mapped to chromosome 17 between the markers D17Mit133 and D17Mit180. This position made theα A-crystallin–encoding gene (Cryaa) an excellent candidate gene. Sequence analysis revealed a mutation of a T to an A at position 371 in the Cryaa cDNA. The mutation was confirmed by an additional MnlI restriction site in the genomic DNA of homozygous mutants leading to replacement of Val with Glu at codon 124 affecting the C-terminal region of theα A-crystallin.

conclusions. The Aey7 mutant represents the first dominant mouse cataract mutation affecting the Cryaa gene. The mutation leads to progressive opacification of the lens. Compared with the β- and γ-crystallin–encoding genes, mutations in theα -crystallin–encoding genes are rare.

The α-crystallins represent the major class of water-soluble proteins in the lens, forming large complexes of approximately 800 to 1000 kDa composed of two subunits, αA- andα B-crystallin. The isolated subunits have molecular masses of 20 and 22 kDa (αA- and αB-crystallin, respectively), and the isoelectric points of the native proteins have been reported to range from pH 4.5 to 5.0. ^{1 2} The α-crystallins are considered to be structural proteins and the subject of a variety of posttranslational modifications (truncation, glycosylation, glycation, carbamylation, and acetylation; for review see Groenen et al. ³ ). 

The two related proteins αA- and αB-crystallin are encoded by two genes, Cryaa and Cryab, which are located on mouse chromosomes 17 and 9, respectively. Both genes have been cloned in a variety of species, including chicken, hamster, human, mouse, rabbit, and rat. Both genes contain three exons of similar size. ⁴ In rodents (mouse, rat, hamster, rabbit) an alternative splice product can be observed in 10% to 20% of the Cryaa transcripts. From intron A an additional 69 base pairs (“insert exon”) are included in the mature mRNA leading to a protein 23 amino acids longer than the usual αA-crystallin, which is referred to as aA^ins-crystallin. ⁵  

The expression of the two Crya genes is not uniform, even if both are expressed at very high levels in the lens. The αA-crystallin can be considered a lens-specific protein, because only trace amounts are found outside the lens in the spleen. ^{6 7} During embryogenesis, Cryaa expression is observed in the lens cup of the rat ⁸ and the mouse at embryonic day (E)10. ⁹ Then, αA-crystallin is present in the posterior half of the lens vesicle, ¹⁰ and later on, it becomes very abundant in lens fiber cells. ⁹  

In contrast, Cryab is expressed ubiquitously. In rat lenses,α B-crystallin can first be detected at the end of embryonic development ¹¹ ; however, in the mouse, it is already present at E9.5. After birth, it is found preferentially in the epithelial cells. ⁹ Transcripts can also be found in marked amounts in brain, heart, skeletal muscle, lung, thymus, and kidney and in a variety of cell lines. ⁷ During embryogenesis, expression of Cryab mRNA was detected by in situ hybridization in the primitive heart of the mouse at E8.5 and in the myotome of the somites at E10.5, supporting the hypothesis that functions of αB-crystallin may be coupled to the activation of genetic programs responsible for myogenic differentiation and cardiac morphogenesis. ¹²  

Because of the expression pattern of the Crya genes, it is thought that several diseases may be caused or accompanied by alterations in the α-crystallins. In the mouse, Brady et al. ¹³ reported the consequence of the loss of the Cryaa gene in homozygous knockout mutants. The only defects produced by the targeted disruption of the Cryaa gene are lens opacities resulting from inclusion bodies containingα B-crystallin. Furthermore, a recessive missense mutation in the Cryaa gene was reported recently as a cause for the lens opacity in the lop18 mouse. ¹⁴ The homozygous mutants exhibit a degeneration of the lens cortex, posterior migration of lens epithelial nuclei, and formation of abnormal lens fibers at the posterior pole resulting in a large white cataract. In a human family with dominant zonular central cataract, a missense mutation in the CRYAA gene leading to replacement of Arg at position 116 with Cys (R116C) was also identified. ¹⁵ A further human cataract, but with recessive inheritance was reported to be caused by the nonsense mutation W9X, resulting in a premature stop codon of CRYAA. ¹⁶  

A mutation in the human αB-crystallin–encoding gene (CRYAB), an A-to-G transition at nucleotide 3787 in the genomic sequence, leads to a replacement of Arg 120 with Gly and to dominant, desmin-related myopathy with cataract formation. ¹⁷ In contrast, the knockout of the Cryab gene in the mouse does not lead to the formation of cataracts, even in the homozygous stage. ¹⁸  

In the course of the analysis of mouse mutants obtained by a large-scale ethylnitrosourea (ENU) mutagenesis program, ^{19 20} we identified several dominant cataract mutations. We report one of them, which was previously referred to as Aey7. It has been mapped to chromosome 17 and finally characterized as the first dominant Cryaa mutation in the mouse. 

Materials and Methods

Mice

Mice were kept under specific pathogen-free conditions at the National Research Center for Environment and Health (GSF) and monitored within the ENU mouse mutagenesis screen project. ^{19 20} The use of animals was in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and with the German Law on Animal Protection. 

Male C3HeB/FeJ mice were treated with ENU (160 mg/kg) at the age of 10 weeks according to Ehling et al. ²¹ and mated to untreated female C3HeB/FeJ mice. The offspring of the ENU-treated mice were screened at the ages of 4 to 6 months for the presence of cataracts with the aid of a slit lamp (SLM30; Carl Zeiss, Oberkochen, Germany). ²² Mice with lens opacities were tested for a dominant mode of inheritance. Homozygotes were obtained by brother-×-sister mating. 

Phenotypic Characterization

The eyes of the mutants were examined continuously during the postnatal life with the slit lamp. For documentation, lenses were enucleated under a dissecting microscope (MZ APO; Leica, Heidelberg, Germany) and photographed. 

Histologic analysis of the eyes was performed at birth and at the ages of 3 and 7 weeks. The eye globes were fixed for 3 hours in Carnoy’s solution and embedded in JB-4 plastic medium (Polysciences, Inc., Eppelheim, Germany), according to the manufacturer’s protocol. Sectioning was performed with an ultramicrotome (OMU3; Reichert-Jung, Walldorf, Germany). Serial transverse 3-μm sections were cut with a glass knife and stained with methylene blue and basic fuchsin. The sections were evaluated with a light microscope (Axioplan; Zeiss). Images were acquired by means of a scanning camera (Progress 3008; Jenoptik, Jena, Germany) and imported into an image-processing program (Photoshop, ver. 5.5 and Adobe Illustrator, ver. 8.0; Adobe, Unterschleissheim, Germany). 

Mapping

Homozygous carriers (first generation) were mated to wild-type C57BL/6J mice, and the offspring (second generation) were backcrossed to the wild-type C57BL/6J mice. DNA was prepared from tail tips of cataractous offspring of the third generation (G3) according to standard procedures. DNA was adjusted to a concentration of 50 ng/μl. For a genome-wide linkage analysis, several microsatellite markers were used for each autosome. ²³  

PCR and Sequencing

For the molecular analysis, RNA was isolated from lenses of C3H/El, T-stock, 129, JF-1, and C57BL/6J wild-type mice and from homozygous mutant mice at the age of 4 weeks. RNA samples from lenses were reverse transcribed to cDNA using a kit (Ready-to-Go; Pharmacia Biotech, Freiburg, Germany); genomic DNA was isolated from tail tips or spleen of wild-type C3HeB/FeJ and C57BL/6J mice or from homozygous mutants, according to standard procedures. For amplification of cDNA from the Cryaa gene, primers were selected from the EMBL and GenBank databases (Table 1 ; EMBL is provided in the public domain by the European Molecular Biology Laboratory, Heidelberg, Germany, and is available at http://www.embl-heidelberg.de; GenBank is provided in the public domain by the National Center for Biotechnology Information and is available at http://www.ncbi.nlm.nih.gov/genbank/). 

PCR was performed using a thermocycler (Clonetech, Heidelberg, Germany, or PE-Biosystems, Weiterstadt, Germany). The annealing temperatures are included in Table 1 . PCR products were analyzed on a 1% agarose gel. Sequencing was performed commercially (SequiServe, Vaterstetten, Germany) immediately after isolation of the DNA from the gel using an extraction kit (Qia-Quick; Qiagen, Hilden, Germany). Some of the PCR products using the primers Cryaa2-L5 and Cryaa2-R2 were digested for 2 hours at 37°C by 5 U restriction endonuclease MnlI (MBI Fermentas, St. Leon-Rot, Germany), and the reaction products were analyzed on a 10% acrylamide gel. 

Computer-assisted predictions of the biochemical properties of proteins were performed using the Proteomics tools of the ExPASy molecular biology server (provided by the Swiss Institute of Bioinformatics, Geneva, and available at no charge to academic users at http://www.expasy.ch). 

Materials

Chemicals were from Merck (Darmstadt, Germany) or Sigma Chemical Co. (Deisenhofen, Germany). The enzymes used for cloning and reverse transcription were from Roche (Mannheim, Germany). 

Results

The Aey7 mutant was detected by slit lamp screening of the offspring of ENU-treated male mice. Progressive opacification of the lens was observed starting as an opacity in the embryonic nucleus and posterior suture at eye opening (postnatal day 12) and terminating as nuclear and zonular opacity at approximately the age of 2 months. The eyes were reduced in size. These defects were more severe in the homozygous mutants than in the heterozygotes (Fig. 1) . 

The histologic analysis did not reveal any abnormalities in the lens structure at the early postnatal stages (postnatal days 1 or 21; data not shown). At the age of 7 weeks, the sections of homozygous mutants exhibited numerous clefts in a subcortical zone and in the fetal nucleus (Fig. 2c) . The topography of the destruction corresponded to that of the opaque regions observed with the slit lamp. Thus, the clefts in the histologic sections were thought to be the consequence of the liquefied lens fibers in the particular zones. As outlined in higher magnifications of the lens bow area (Figs. 2b 2d) , no differences could be observed between the wild-type and the homozygous mutants in this region of the lens. The epithelium and also the newly differentiating lens fibers were well organized, and the nuclei were arranged in a wavelike manner. 

A mutant line was established. The mutation had a complete penetrance, and the litter size in the matings of heterozygotes as well as of homozygotes indicated normal fertility and viability of the mutants. Thirty cataractous mice from the backcross (G3) were used for genome-wide mapping. The results indicated linkage with markers on mouse chromosome 17, and the following gene order was observed: D17Mit133 (10.0 ± 5.5 centimorgans [cM]), Aey7 (3.3 ± 3.3 cM), D17Mit50, D17Mit180 (10.0 ± 5.5 cM), D17Mit20. These results are in good agreement with the current report of the Chromosome Committee for mouse chromosome 17 (provided by Jackson Laboratories, Bar Harbor, ME, and available at http://www.informatics.jax.org/bin/ccr/). A detailed haplotype analysis is given in Figure 3a and the partial map of mouse chromosome 17 in Figure 3b . 

Based on this mapping information, the Cryaa gene (17.4 cM from the centromere) was considered to represent a good candidate gene for causing this type of cataract. The corresponding cDNA was amplified both in the wild-type mice and in the heterozygous mutants using cDNA templates derived by reverse transcription of RNA from lenses of 4-week-old mice. 

Within the coding sequence, several polymorphic sites were observed in our C3H/El colony compared with the database (accession no. J00375 for the 5′ part and J00376 for the 3′ end); however, all predicted amino acids do not alter the primary sequence of the protein. The entire cDNA from wild-type C3H/El mice (including the insert exon) has been added to the EMBL database (accession no. AJ310308). 

The only sequence alteration cosegregating with the mutant phenotype was a replacement of T with A at position 371 (exon 3) of the Cryaa gene, counting the first base in the ATG start codon as base pair 1. The insert exon, which is spliced into the mature mRNA of only 10% of the rodent Cryaa transcripts, was not considered in this calculation (Fig. 4) . The mutation leads to the appearance of a new MnlI restriction site in the mutant mice, but is not present in the wild-type sequence of C3H/El and several other mouse strains. The cosegregation of the mutation was validated by the presence of the MnlI restriction site in five homozygous mutants (Fig. 5) . Therefore, the new allele should be referred to as Cryaa ^Aey7 . 

The mutation is predicted to lead to a Val→Glu exchange at codon 124, counting the Met start codon as 1. To allow comparison across species borders, the amino acids encoded in rodents by the insert exon are not considered. At this position (124), Glu occurs in none of the knownα -crystallins from 28 mammalian species, from chicken and frog (which has Leu instead of Val at this position), or from 13 primates. ^{24 25} The ProSite scan of ExPASy (http://www.expasy.ch) does not reveal changes at this particular site; particularly, the recognition sequence for the phosphorylation site at Ser122 is not affected. The ProtScale analysis of ExPASy, looking for hydrophobic or hydrophilic regions within the protein, revealed also only a very slight difference in the region surrounding position 124. 

Discussion

A new dominant cataract mutation was observed among the F₁ offspring of ENU-treated male mice. This opacity, preliminarily referred to as Aey7, was demonstrated to be caused by a T→A exchange within the third exon of the Cryaa gene, and the mutant allele was therefore designated Cryaa ^Aey7 . The predicted amino acid in the mutants is Glu instead of Val at position 124, close to the C terminus. The lens opacity is already visible at eye opening. The cataractous changes coincide with the expression of the Cryaa gene, which is observed first in the lens cup. Later on, αA-crystallin becomes very abundant in lens fiber cells ^{8 9 10 11} and is considered as a major structural protein of the ocular lens. ²  

α-Crystallin participates in the intracellular architecture through cellular filaments. Together with CP49, a lens-specific cytoskeletal protein, α-crystallins form the beaded filaments. ²⁶ α A-Crystallin also interacts with tubulin ²⁷ and actin. ²⁸ Moreover, there are several lines of biochemical evidence that α-crystallin may become associated with the plasma membrane. Because the molecular sites of these interactions are not yet known in detail, ²⁹ whether the V124E mutation described herein is involved in these interactions is open to speculation. 

Besides its structural properties, αA-crystallin is the target of posttranslational alterations, and one of its most important modifications is phosphorylation. Past studies have demonstrated that the major site of αA-crystallin in vivo phosphorylation is Ser122, which is phosphorylated in a cAMP-dependent manner. ³⁰ This phosphorylated form of αA-crystallin could be detected in human lenses only from adolescent, adult, and senile donors, but not in infants, suggesting a developmental regulation of this particular kind of modification. ³¹ Computer-assisted prediction programs, such as ProSite (ExPASy), did not support the hypothesis that the V124E mutation influences the use of the very close phosphorylation site at Ser122. 

However, the most exciting finding concerns the function ofα A-crystallin as a molecular chaperone. ³² α A-Crystallin prevents thermal aggregation of several enzymes and even of β- and γ-crystallins. Chaperone activity is essential for the lens, because degradation and extrusion of defective proteins is not possible as it is in other tissues. Moreover, the lens is exposed to a variety of damaging agents, particularly in light of various wavelengths leading to oxidative effects on quite a number of lens proteins. α-Crystallin has a substrate specificity different from other chaperones and recognizes specific nonnative intermediates formed during denaturation only. ³³  

Based on the gene structure, Wistow ³⁴ thought the overall structure of α-crystallins to consist of a globular N-terminal domain of two symmetry-related motifs and a somewhat longer C-terminal domain also consisting of two motifs. The two globular domains, which are built up by two exons, are fused by a short connecting peptide, which is extended in the rodent αA^ins-crystallin by 23 amino acids ⁵ and has a significant reduced chaperone activity compared with the normal αA-crystallin. ^{35 36} The C terminus of the αA-crystallin consists of two rather hydrophilic domains ² that are exposed to the surface and tend to form tetrameric assemblies. ³⁷ Moreover, there is considerable evidence for the involvement of the flexible C-terminal extension in chaperone activity. Its truncation or immobilization greatly reduces this capacity, suggesting that the hydrophilic tail is likely to be important in keeping complexes of chaperones and bound proteins in solution. ⁴ The chaperone activity ofα -crystallin was first localized to amino acid residues 158-173 of the C-terminal region of αA-crystallin, ³⁸ but recently, reduced chaperone-like activity was demonstrated also in anα A-crystallin mutation (Asp69Ser) ³⁹ and by the introduction of a hydrophobic tryptophan at position 172. ⁴⁰  

The mutation described in this article replaces a neutral, hydrophobic amino acid (Val) with an acidic amino acid (Glu) in the C-terminal part (position 124) of the αA-crystallin. The same region of theα A-crystallin is affected by a mutation in human CRYAA causing a dominant zonular central cataract phenotypically very similar to Aey7. It was characterized by a missense mutation gene leading to replacement of Arg with Cys at position 116 (R116C). ¹⁵ Detailed biochemical analysis of this mutant protein demonstrated a fourfold reduction in chaperone-like activity, but it tends to bind to membranes 10 times more than the wild-type form. ⁴¹ Similar explanations may be suggested for cataractogenesis in the Cryaa ^Aey7 mutants. 

In mouse and human, a few other mutations in the Cryaa/CRYAA genes have already been described. The loss of the entire Cryaa gene in mouse knockout mutants leads to an opacification of the lens only in homozygous conditions. ¹³ In the recessive lop18 mouse mutants, a missense mutation in the first exon of the Cryaa gene converting codon 54 Arg to His was demonstrated recently as cause for the phenotype. ¹⁴ A recessive cataract has been also reported in human, which is caused by the nonsense mutation W9X, resulting in a premature stop codon of CRYAA. ¹⁶  

Of interest and in contrast to the other abundant lens proteins, theβ - and γ-crystallins, mutations in the Cryaa and CRYAA genes lead to both recessive and dominant phenotypes. Mutations affecting the N-terminal part or even the loss of the entire protein result in recessive phenotypes, whereas mutations in the C-terminal part leads to dominant phenotypes. Most likely, the dominant mutation inhibits interactions with other proteins affecting the chaperone activity, affinity with the membranes, or interactions with the lens intermediate filaments. 

 Present affiliations: ⁴Institute of Human Genetics, University of Erlangen-Nürnberg, Germany; and the ⁶German Research Center for Biotechnology (GBF), Braunschweig, Germany.

 Supported by Grant 01KW9610/1 from the German Human Genome Project (RB, EW, MHdA).

 Submitted for publication April 19, 2001; revised July 23, 2001; accepted August 1, 2001.

 Commercial relationships policy: C, I (RB, MHdA); N (all others).

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked“ advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

PCR Primers with Annealing Temperatures and Length of the Products

Designation	Lab-Number	Sequence (5′ → 3′)	Accession Number	Annealing Temperature (°C); Fragment Size (bp)
Cryaa-L1	30553	TCC TGT CTG ACT CAC TGC CAG CC	J00375	50–55
Cryaa-R1	30554	AAG CCA CCT TCT CAG ACC CTC AGC	J00376	792
Cryaa-L2	32175	GCC CCA AAT GCA GGC TCT AGC	J00375	58
Cryaa-R5	32178	TGA TCA TTT CTA ATC CCC TGT CAC CC	J00375	1124
Cryaa-L3	32176	CCT CCT GTG CTG TGC TGG CC	J00375	58
Cryaa-R5	32178	TGA TCA TTT CTA ATC CCC TGT CAC CC	J00375	159
Cryaa-L4	32179	GGT CCG ATC TGA CCG GGA C	J00375	55
Cryaa-R2	31906	TCT CGC TGT GGC CAG CAT CC	J00376	291
Cryaa-L5*	32864	CAT GGC TAC ATT TCC CGT GAA TTT C	AJ310308	58
Cryaa-R2	31906	TCT CGC TGT GGC CAG CAT CC	J00376	150

*  Underlined base represents one of the polymorphic sites in mouse Cryaa genes.

Figure 1.

 

Gross appearance of lenses of the Aey7 mutant. Lenses of a heterozygous (left) and a homozygous (right) Aey7 mutant at the adult stage. Both types of mutants exhibited a nuclear cataract and zonular opacity in a subcortical region. The lens of the homozygous Aey7 mutant was smaller than in the heterozygotes and displayed a more pronounced nuclear cataract.

Figure 2.

 

Histologic analysis of lenses of the Aey7 mutant. Sections through lenses of 7-week-old wild-type (a, b) or homozygous mutant mice (c, d) are shown. The mutants showed numerous clefts in the lens core and in the subcortical region (c). The age-matched control did not show abnormalities. Magnification of the lens bow region demonstrated the regular arrangement of the epithelial cells and the novel secondary fiber cells as well as the presence of cell nuclei in a well-shaped order, both in the wild-type (b) and in the homozygous mutant (d). C, cornea; LB, lens bow; LE, lens epithelium. Scale bars, 100 μm.

Figure 3.

 

Linkage analysis of the Aey7 mutation localized on chromosome 17. (a) Heterozygous mutant mice were outcrossed to wild-type C57BL/6J mice, the heterozygous carriers were backcrossed to wild-type C57BL/6J mice. Among the offspring, only the cataractous mice were analyzed for their parental genotypes with respect to a variety of microsatellite markers; results are shown for those on chromosome 17. The total number of progeny scored for each locus is given on the right of the boxes, including the calculated distances between the loci (in centimorgans). The number of progeny that inherited each haplotype is given below the boxes. (b) A partial chromosome map shows the location of the Aey7 mutation in relation to relevant markers and to the candidate gene Cryaa. Numbers to the left of the chromosome indicate the genetic distance in centimorgans from the centromere, according to the 2000 Chromosome Committee report (Jackson Laboratories). At right, the actual linkage data are summarized.

Figure 4.

 

Characterization of the Aey7 mutation within the Cryaa gene. The genomic structure of the mouse Cryaa gene is demonstrated; the exon boundaries are marked according to King and Piatigorsky ⁵ and Jaworski. ²⁵ The counting of nucleotides and amino acids is given excluding the insert exon within intron 1 and the corresponding amino acids. A part of the cDNA sequence of the mouse Cryaa gene is given (EMBL, accession no. AJ310308) and the corresponding deduced amino acid sequence is shown above the cDNA. The mutation is indicated in bold and shaded in gray; with the changed amino acid shown below the sequence. The new MnlI restriction site at position 370-373 is underlined.

Figure 5.

 

Cryaa digest by MnlI. The genomic DNA of the Cryaa gene was amplified. The PCR fragment was analyzed by agarose gel electrophoresis with (+) and without (−) subsequent digest by MnlI. The fragment from the wild-type C3H/El, C57BL/6J (C57BL), T-stock (T), 129, and JF-1 (JF) mice could not be digested (a), but the cDNA from lenses of several male (subjects 156, 158, 159) and female (subjects 157, 160) homozygous Aey7 mutants led to the expected digest pattern (b).

The authors thank Ingenium Pharmaceutical, AG, Martinsried, Germany, for kindly providing the Aey7 mutant; Francoise André, Gerlinde Bergter, Erika Bürkle, Nicole Hirsch, Sabine Manz, Andreas Mayer, Dagmar Reinl, and Monika Stadler for expert technical assistance; and Utz Linzner, GSF-Institute of Experimental Genetics, Neuherberg, Germany, for providing the oligonucleotides.