Skip Navigation


Human Molecular Genetics Advance Access originally published online on March 11, 2004
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
13/9/945    most recent
ddh110v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (16)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Reddy, M.A.
Right arrow Articles by Slingsby, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Reddy, M.A.
Right arrow Articles by Slingsby, C.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Human Molecular Genetics, 2004, Vol. 13, No. 9 945-953
DOI: 10.1093/hmg/ddh110
Human Molecular Genetics, Vol. 13, No. 9 © Oxford University Press 2004; all rights reserved

Characterization of the G91del CRYBA1/3-crystallin protein: a cause of human inherited cataract

M.A. Reddy1,*, O.A. Bateman2, C. Chakarova1, J. Ferris3, V. Berry1, E. Lomas4, R. Sarra2, M.A. Smith2, A.T. Moore1, S.S. Bhattacharya1 and C. Slingsby2

1Department of Molecular Genetics, Institute of Ophthalmology, 11–43 Bath Street, London EC1V 9EL, UK, 2Department of Crystallography, Birkbeck College, Malet Street, London WC1E 7HX, UK, 3Department of Ophthalmology, Cheltenham General Hospital, Cheltenham, UK and 4Human Genome Mapping Project Resource Centre, Babraham, Cambridge, UK

Received January 5, 2004; Revised February 18, 2004; Accepted March 3, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Congenital cataract is a leading cause of visual disability in children. Inherited isolated (non-syndromic) cataract represents a significant proportion of cases and the identification of genes responsible for inherited cataract will lead to a better understanding of the mechanism of cataract formation at the molecular level both in congenital and age-related cataract. Crystallins are abundantly expressed in the developing human lens and represent excellent candidate genes for inherited cataract. A genome-wide search of a five-generation family with autosomal dominant lamellar cataract demonstrated linkage to the 17p12–q11 region. Screening of the CRYBA1/3 gene showed a 3 bp deletion, which resulted in a G91del mutation within the tyrosine corner, that co-segregated with disease and was not found in 96 normal controls. In order to understand the molecular basis of cataract formation, the mutant protein was expressed in vitro and its unfolding and refolding characteristics assessed using far-UV circular dichroism spectroscopy. Defective folding and a reduction in solubility were found. As the wild-type protein did not refold into the native conformation following unfolding, a corresponding CRYBB2 mutant was genetically engineered and its refolding characteristics analysed and compared with wild-type CRYBB2. Its biophysical properties support the hypothesis that removal of the glycine residue from the tyrosine corner impairs the folding and solubility of ß-crystallin proteins. This study represents the first comprehensive description of the biophysical consequences of a mutant ß-crystallin protein that is associated with human inherited cataract.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The term cataract refers to opacification of the ‘crystalline’ lens in the human eye. Cataract is the most common cause of visual loss in humans (1) and may be broadly divided into early-onset (congenital or juvenile) and age-related cataract. Although congenital cataract is much less common than age-related cataract, it is still responsible for approximately one-tenth of childhood blindness worldwide (2). A third of children with isolated cataract (and without other ocular and systemic anomalies) have inherited cataract and the most common form of inheritance is as an autosomal dominant trait (3). Isolated autosomal dominant cataract (IADC) is genetically heterogeneous and to date 14 genes and nine additional loci have been implicated in human IADC (4). The water-soluble lens crystallins account for nearly 90% of the total lens proteins and, not surprisingly, mutations in the crystallin genes represent a large proportion of the mutations identified in IADC to date (4).

Lens crystallins belong to two protein families, the {alpha}- and ß{gamma}-crystallins (5).The main function of these highly expressed proteins is to engage in tight packing as this provides both transparency and refraction (6) with solubility and stability being their key biophysical properties (7). In order to enhance transparency, the bulk of the lens cells have lost their cellular machinery for macromolecular synthesis and protein degradation. High levels of {alpha}-crystallin, with chaperone-like activity, are present in the lens and bind to substrate proteins, including the ß{gamma}-crystallins, to prevent their precipitation (8). However, if abnormal proteins accumulate they are likely to cause fluctuations in refractive index (9) and lead to loss of transparency of the lens. Mutations can alter the structure of synthesized proteins in several ways: for example, in the case of monomeric {gamma}-crystallins the protein may be unable to fold and aggregate (10), the crystallin may fold but be of lower thermal stability (11), or the crystallin may fold but be of lower solubility (12,13). For oligomeric proteins comprising different gene products, the impact on structure may also be on subunit assembly.

The presence of {alpha}- and ß{gamma}-crystallins within the lens makes their encoding genes excellent candidates for IADC. Mutations have been found in the genes coding for these proteins (4) and reflect the role these proteins have in the development of the lens (14). Mutations associated with IADC have been described in the basic ß-crystallin genes CRYBB1 and CRYBB2 (15–18).

Two different splice site mutations in the CRYBA1/3 gene at exactly the same position have been described in two unrelated families and cause different cataract phenotypes (19). An Indian family with dominantly inherited nuclear sutural cataract was found to have a co-segregating sequence change at the donor splice site for intron 3 (G474A) (20). A Brazilian family, with autosomal dominant pulverulent cataract with posterior sutural opacities, was found to have a different co-segregating mutation at the same site (G474C) (21). Functional studies on the G474A mRNA show that the first and second Greek key motifs encoded by exons 3 and 4 are absent in the mutant G474A polypeptide (22).

In this paper, we report the results of a molecular genetic study of a five-generation British family with autosomal dominant lamellar cataract. We have identified a co-segregating G91del mutation in the CRYBA1/3 gene and analysis of the mutant and a corresponding CRYBB2 mutant in vitro demonstrates defective folding and a reduction in solubility. This study represents the first comprehensive description of the biophysical consequences of a mutant ß-crystallin protein that is associated with human inherited cataract.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Clinical findings
Members of a five-generation family (Fig. 1) from the southwest of the UK were invited to take part in this research. All affected individuals displayed bilateral lamellar cataracts with variable severity (Fig. 2). The cataracts were inherited as an autosomal dominant trait. The clinical details are outlined in Table 1.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 1. Haplotype analysis of pedigree U.

 


View larger version (104K):
[in this window]
[in a new window]
  		Figure 2. An annotated image of the lens in individual V : 11 in pedigree U.

 

View this table:
[in this window]
[in a new window]
  		Table 1. Clinical findings for pedigree U
 
Linkage analysis and mutation screening
Genomic-wide linkage analysis was performed using DNA from 10 affected and five unaffected individuals. Significantly positive two-point LOD scores were obtained for the markers D17S1857 (Zmax=3.32 at q=0) and D17S1824 (Zmax=3.09 at q=0) on 17q11. Subsequent genotyping (Fig. 1) confirmed linkage to a genetic interval of at least 23 cM from 17p12–q11 (Table 2). Screening of the candidate CRYBA1 gene in this interval identified a 3 bp deletion mutation in exon 4 (Fig. 3) that co-segregated with the disease. This change results in the deletion of a glycine amino acid (G91del). This sequence change was not found in 96 normal controls.


View this table:
[in this window]
[in a new window]
  		Table 2. LOD scores for family U using markers from 17p11 to 17q11
 


View larger version (10K):
[in this window]
[in a new window]
  		Figure 3. (A) Wild-type genomic DNA in CRYBA1 and (B) mutant DNA with predicted amino acid sequences. The deleted nucleotides are shown within the box.

 
Solubility of wild-type and mutant CRYBA3–crystallin proteins.
When recombinant wild-type and mutant CRYBA3–crystallins were expressed in E. coli, SDS–PAGE gels showed that the wild-type protein was expressed in the soluble fraction whereas the mutant was found in the insoluble pellet. The pellet was solubilized using ‘unfolding conditions’ which were also used for anion-exchange chromatography (see Materials and Methods).

Purified wild-type CRYBA3–crystallin, in 20 mM sodium phosphate, pH 7.5, 1 mM DTT, could be concentrated to more than 100 mg ml–1 at 22°C. However, when the mutant CRYBA3–crystallin, purified from the insoluble pellet under dissociating conditions, was exchanged into native solution conditions (by dilution into 0.6 M urea, 20 mM sodium phosphate, pH 7.5, 1 mM DTT), it could not be concentrated above ~100 µg ml–1. In fact most efforts to handle the mutant crystallin resulted in its loss, presumably due to its sticking to surfaces. The low solubility of the purified mutant polypeptide in comparison to the wild-type is consistent with its presence in the insoluble pellet following bacterial expression.

Size estimation of mutant CRYBA3–crystallin compared with wild-type
Wild-type CRYBA3–crystallin behaves as a dimer when chromatographed by gel permeation chromatography at pH 7.5 (Fig. 4). However, the mutant protein, following rapid removal of urea on a desalting column, eluted in the void volume of the gel filtration column (Fig. 4), indicating it had a molecular weight higher than 300 kDa.



View larger version (10K):
[in this window]
[in a new window]
  		Figure 4. Gel permeation chromatography of mutant and wild-type CRYBA3 on Superose 12 HR 10/30. The native wild-type CRYBA3 elutes with an elution volume (Ve) of 12.5 ml, indicating that it is a dimer in solution. The mutant, refolded from urea, eluted with a Ve of 7.1 ml, which is the void volume of the column, indicated by the arrow. The sample of wild-type CRYBA3, which had been unfolded in urea and then refolded, also eluted at the void volume.

 
Secondary structure determination of mutant CRYBA3–crystallin compared with wild-type
The polypeptide secondary structure was estimated by far-UV circular dichroism (CD) spectroscopy under native conditions, namely 20 mM sodium phosphate, pH 7.5. The wild-type CRYBA3–crystallin gave a far-UV CD spectrum (Fig. 5A, brown spectrum) similar to other ß{gamma}-crystallins and typical of ß-sheet proteins except that the spectrum has features in the 220–230 nm region which are likely due to tryptophan side chains (23). The ratio of the signals at around 195–215 nm indicates that the protein is well folded. In the presence of 6 M urea the CD spectrum of wild-type CRYBA3–crystallin shows the characteristic of an unfolded polypeptide chain (Fig. 5A, green spectrum). When the CD spectrum of the deletion mutant of CRYBA3 was recorded in 0.6 M urea, only a limited region of the spectrum (205–260 nm) was available due to absorption by urea; however, the spectrum clearly showed evidence of secondary structure (data not shown). In order to extend the measurement further into the ultraviolet, the mutant CRYBA3–crystallin was rapidly buffer-exchanged into folding conditions by passing the sample through a desalting column whereby urea was eliminated. The mutant polypeptide could now be recorded over the same wavelength range as the wild-type protein. The spectrum of mutant CRYBA3 (Fig. 5A, red spectrum) shows that, as judged by the strength of signal at 215 nm, the mutant appears to have a greater content of ß-strand secondary structure than the wild-type, lacks the detail in the 220–230 nm region, and the 195/215 ratio is quite distinctive from that of wild-type CRYBA3–crystallin. In sum, the refolded structure of the deletion mutant of CRYBA3–crystallin has changed considerably from the wild-type native structure.



View larger version (16K):
[in this window]
[in a new window]
  		Figure 5. (A) Far-UV circular dichroism spectra of CRYBA3–crystallins; (B) far-UV circular dichroism spectra of CRYBB2–crystallins.

 
Unfolded/refolded wild-type CRYBA3–crystallin
Although the mutant and wild-type proteins have secondary structure, their CD spectra are clearly different, and the proteins differ both in size and in their solubility under native solution conditions. However, as the purification protocol necessitated the use of dissociating/unfolding conditions in order to solubilize the mutant polypeptide from the insoluble pellet, it was necessary to address the question of what happens when the wild-type protein is subjected to equivalent unfolding and refolding conditions. When the wild-type CRYBA3–crystallin was unfolded and then refolded rapidly into a urea-free medium, the CD spectrum (Fig. 5A, blue spectrum) showed that the protein did not refold into the native structure. The spectrum of refolded wild-type CRYBA3–crystallin indicated a ß-strand secondary structure content that was intermediary between that of the native wild-type and the refolded mutant, whereas the 195/215 ratio resembled the refolded deletion mutant more closely than that of the wild-type native spectrum. On gel permeation chromatography, the unfolded/refolded wild-type CRYBA3 eluted largely in the void volume, although some dimer was recovered (Fig. 4), and thus by this technique behaved more like the mutant polypeptide.

The results so far show that wild-type CRYBA3–crystallin is not able to refold to a native dimeric structure following unfolding in 6 M urea. However, this refolded non-native state, like the refolded mutant, is rich in ß-strand secondary structure conformation. It is possible that the overexpressed mutant protein in the lens forms these low-solubility high-molecular-weight aggregates that are rich in ß-strand secondary structure. However, we are unable to confirm in vitro that the main difference between mutant and wild-type CRYBA3 is the tendency to form such aggregates, as wild-type CRYBA3–crystallin cannot be refolded into a native conformation, and the deletion mutant can only be studied in a refolded form.

In order to confirm the idea that deletion of the conserved glycine is responsible for causing major disruption of the ß{gamma}-crystallin domain fold, it was necessary to repeat the deletion on a ß-crystallin for which the unfolding/refolding transition is reversible. As the glycine residue is so conserved in the lens ß{gamma}-crystallin family (Fig. 6), the effect of its deletion on the integrity of the domain fold was ascertained by repeating the equivalent mutation on human CRYBB2–crystallin. It was anticipated that human CRYBB2–crystallin would reversibly unfold as the three-dimensional structure of the bovine lens orthologue was solved from crystals grown from the refolded protein (24) and the unfolding/refolding transition for the rat orthologue was shown to be reversible (25).



View larger version (29K):
[in this window]
[in a new window]
  		Figure 6. The sequence alignments of the tyrosine corner region in the N-terminal domain of the human ß{gamma}-crystallin family aligned with the corresponding region from bovine ßB2- and {gamma}-crystallins alongside a ribbon representation of a typical ß{gamma}-crystallin domain. The conserved glycine is highlighted in pink with its domain location indicated by the pink arrow, and the conserved tyrosine is highlighted in royal blue in the alignment and is appended to the domain. Residues highlighted in blue are in ß-strand secondary structure.

 
Characterization of the corresponding glycine deletion mutation in CRYBB2–crystallin
When the recombinant mutant CRYBB2–crystallin with G76 deletion, topologically equivalent to the G91 deletion in CRYBA1/3, was expressed in E. coli, the protein was located in the insoluble pellet. The pellet was solubilized using ‘unfolding conditions’ which were also used for anion-exchange chromatography to purify the mutant. Low levels of protein in solution were obtained by diluting the mutant CRYBB2–crystallin into native buffer ‘folding conditions’. Attempts were made to chromatograph the ‘refolded’ mutant by ion-exchange chromatography, although it behaved anomalously on the column by not resolving into discrete protein peaks. The mutant CRYBB2–crystallin was obtained free of chemical denaturants and in the presence of 20 mM sodium phosphate, pH 7.5 could be concentrated to around 4 mg ml–1 at 22°C. This is in contrast to the wild-type CRYBB2–crystallin, which, regardless of whether it has been refolded or not, shows high protein solubility with solutions of more than 50 mg ml–1 being routinely used for crystallization. The mutant CRYBB2 polypeptide secondary structure was estimated by far UV circular dichroism (CD) spectroscopy under native buffer conditions, namely 20 mM sodium phosphate, pH 7.5. The spectrum of the urea-free mutant CRYBB2–crystallin is that of a largely unfolded polypeptide, although it is not a random coil (Fig. 5B, red spectrum). In contrast, the unfolded/refolded wild-type CRYBB2 (Fig. 6B, blue spectrum) is similar to the native wild-type CRYBB2–crystallin (Fig. 5B, brown spectrum).

These data are consistent with wild-type human CRYBB2–crystallin showing reversible unfolding–refolding behaviour. This protein is thus a model system for testing the effects of a mutation to a conserved residue that results in its expression in an insoluble form in E. coli. When the conserved glycine residue in CRYBB2 is deleted, the protein is unable to fold properly and loses solubility, although the resulting non-native polypeptide is more soluble than that of the equivalent CRYBA3–crystallin mutant.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Sequence and three-dimensional structural comparisons show that all the lens ß{gamma}-crystallin polypeptides fold into an N-terminal domain comprising two similar consecutive Greek key motifs 1 and 2, and a similar C-terminal domain comprising Greek keys motifs 3 and 4 (7). Each Greek key motif comprises four consecutive ß-strands (a–d) that intercalate to form two ß-sheets (Fig. 6). The G91 deletion in CRYBA/3 identified in our family with IADC results in loss of a glycine residue from the edge of strand c in the second motif of the N-terminal domain affecting a conserved structural feature known as a tyrosine corner that stabilizes the connection between b and c ß-strands (Fig. 6). Tyrosine corners are present in most ß-sandwich domains where they appear to stabilize the fold topology (26). The alignment of the connecting region between b and c ß-strands of the human ß{gamma}-crystallin sequences with the structural alignment derived from the X-ray structures of bovine {gamma}B and ßB2-crystallins shows that the glycine and tyrosine residues are absolutely conserved in the N-terminal domain (Fig. 6), with corresponding residues in the C-terminal domain showing some minor sequence variations.

The removal of a glycine residue from a highly conserved structural domain is likely to cause major destabilization of the native protein fold. This is in keeping with its cellular location following expression in E. coli where it was found in the insoluble pellet. The mutant protein could be readily solubilized under denaturing conditions, but attempts to refold the protein resulted in high molecular weight aggregates of very low water solubility. Thus the mutation has destroyed the key crystallin attribute of water solubility. These high molecular weight aggregates were, however, found to contain considerable levels of ß-strand secondary structure, and in this sense they had ‘refolded’. As the wild-type CRYBA3–crystallin did not refold into the native conformation following unfolding, it was difficult to show that the mutant refolds in an abnormal manner compared with wild-type.

In order to test the hypothesis that the CRYBA3 mutant refolds in an abnormal manner, the corresponding glycine deletion mutation was made in CRYBB2 and the recombinant protein characterized. The mutant CRYBB2–crystallin was also expressed in an insoluble form. Attempts to refold this mutant protein resulted in a polypeptide of greater water solubility than the corresponding CRYBA3–crystallin mutant, although solubility was still greatly reduced compared with wild-type CRYBB2–crystallin. The CD spectrum of the reduced solubility CRYBB2–crystallin mutant was shown to be largely unfolded, thus formally proving that the glycine residue in a tyrosine corner is essential for a properly folded ß-crystallin.

Based on solution behaviour, involving dynamic subunit-exchange between basic and acidic ß-crystallin homodimers, it is likely that in the normal lens wild-type CRYBA3–crystallin will form heterotypic interactions with basic ß-crystallins (27). A mutation in one CRYB–crystallin gene will also alter the overall profile of assembled ß-crystallin oligomers in the lens. The expression of the mutant protein would be predicted to have a deleterious effect on crystallin oligomer assembly and could contribute to loss of transparency.

Recently, a Swiss family with nuclear cataract was found to have a 3 bp deletion in CRYBA1 that resulted in the same G91del mutation (28). Evidently other factors must contribute towards the determination of the phenotype observed in both of these families with the identical mutation (28). It is not known if the CRYBA3–crystallin expressed in the lens of patients carrying this mutation has a similar secondary structure to the refolded mutant protein described here. However, these experiments do show that this mutant crystallin has retained the propensity to form secondary structure but has limited water solubility. The consequences to the lens could depend on the level of expression, the ability of the newly formed cell to degrade it, the availability of the chaperone {alpha}-crystallin to potentially bind and solubilize it, and the level of general disturbance to the other ß-crystallin components who have reduced levels of a partner.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Clinical examination
Local ethical committee approval was granted and informed consent was obtained from all individuals for clinical and genetic examinations. All members of the family were examined independently by two ophthalmologists (M.A.R. and J.F.). Slit-lamp examination took place on all individuals. DNA was extracted from venous blood for genetic analysis.

Linkage analysis and screening
Linkage analysis by PCR-based microsatellite marker genotyping was used to identify the disease locus. Genomic-wide linkage mapping, using fluorescent labelled dinucleotide microsatellite markers which were on average 10 cM apart in the human genome, was performed at the Linkage Hotel, UK Human Genome Mapping Project Resource Centre, Babraham, Cambridgeshire, UK. The markers were derived from the ABI Prism Linkage Mapping Set Version 2 (Perkin Elmer, Foster City, CA, USA) and the PCR products were analysed using an ABI Prism 3100 DNA Sequencer. Two-point LOD scores were calculated using Genetic Linkage User Environment (GLUE), Human Genome Mapping Project and the MLINK program within Cyrillic 2.1.1 (29). An autosomal dominant model with equal allele frequencies, a gene frequency of 10–4 and a mutation rate of 10–6 was used in calculating the two-point LOD scores.

Following the identification of the likely genetic interval, six primers that flanked each of the six exons of CRYBA1 were used to sequence the coding region. Automated DNA sequencing was performed using an ABI 3100 DNA sequencer (Perkin Elmer, Foster City, CA, USA).

Construction of expression plasmids
The CRYBA1/3 and CRYBB2–crystallin coding sequences, isolated by PCR from a cDNA library of fetal human lens RNA and cloned into pET3a, were a gift from Dr Nicolette Lubsen. In order to introduce a deletion mutation (G91del) in CRYBA1/3, the following oligonucleotide primers were made: 5'-AGTTTATCCTGGAGAGAGAATACCCTC-3' as the forward primer and 5'-GAGGGTATTCTCTCTCCAGGATA AAC-3' as the reverse primer. A deletion mutation in CRYBB2 was introduced using 5'-GTGTTTGAGAAGGA GTACCCCCGCTG-3' as the forward primer and 5'-CAGCGGGGGTACTCCTTCTCAAACAC-3' as the reverse primer. All primers were synthesized by Genosys. Mutagenesis was performed with QuikChange site-directed mutagenesis kit from Stratagene. The plasmid DNA obtained after mutagenesis was sequenced with the T7 promoter primer and was found to contain the desired mutation but no other sequence changes. The mutant proteins were expressed in Escherichia coli and purified as described below.

Expression of recombinant proteins
The pET3a recombinants were transformed into BL21(DE3)pLys or into the Rosetta pLys strain (Invitrogen) and grown and induced as described previously (27,30). Cells were harvested by centrifugation and resuspended in 10 ml 25 mM Tris–HCl, 5 mM EDTA, 50 mM glucose pH 8.0 containing a protease inhibitor (Pefabloc, Merck). Cells were lysed by sonification (3x45 s), DNaseI was then added to 50 µg ml–1 final concentration and the lysate was incubated for 30 min at room temperature. The lysate was then cleared by centrifugation for 30 min at 18 000 rpm. In the case of cells expressing wild-type CRYBA3 and wild-type CRYBB2–crystallins the supernatants were stored at –20°C and in the case of the corresponding mutants the supernatants were discarded and the pellets stored frozen at –20°C. SDS–PAGE gels showed that the wild-type proteins were expressed in the soluble fractions whereas the glycine deletion mutants were found in the insoluble pellets.

Purification of wild-type CRYBA3–crystallin and wild-type CRYBB2–crystallin
The wild-type CRYBA3–crystallin was purified from soluble E. coli proteins by chromatography in ‘native buffers’, using anion-exchange chromatography, followed by cation-exchange chromatography as described previously (27). The bacterial lysate was dialysed overnight at 4°C in 25 mM Tris–HCl, pH 7.5, 1 mM DTT (buffer 1A). The lysate was loaded onto a HiPrep 16/10 Q Sepharose FF column equilibrated with buffer 1A and run at 5 ml min–1. A gradient of the running buffer with 1 M NaCl (buffer 1B) of 0–70% over 10 column volumes was applied and the CRYBA3–crystallin eluted at around 20% buffer 1B. The CRYBA3–crystallin was passed through a desalting column equilibrated with 50 mM MES, pH 5.8, 1 mM DTT (buffer 2A) before being loaded onto a Mono S cation exchange column equilibrated with buffer 2A. When a gradient of this buffer with 1 M NaCl (buffer 2B) of 0–15% over 10 column volumes was applied, the CRYBA3–crystallin eluted at around 6.5% (buffer B). The peak fractions were pooled and the protein concentration was determined using an extinction coefficient of 2.6 for a 1 mg ml–1 solution. The wild-type CRYBB2–crystallin was purified from the bacterial supernatant, as described by Bateman and coworkers (30). The protein concentration was determined using an extinction coefficient of 1.7 for a 1 mg ml–1 solution.

Purification of mutant CRYBA3–crystallin and mutant CRYBB2–crystallin
The pellet of insoluble material from the mutant CRYBA3–crystallin bacterial lysate was dissolved in 25 mM Tris–HCl, pH 7.5, 8 M urea. SDS–PAGE gels indicated that a 10 kDa protein was contaminating the 25 kDa mutant and so the dissolved material was chromatographed on a Mono Q HR 10/10 under dissociating conditions with 25 mM Tris–HCl, pH 7.5, 1 mM DTT, 6 M urea as buffer 3A and a gradient of buffer 3B (buffer 3A/1 M NaCl) of 0–15% over 10 column volumes. The 25 kDa crystallin polypeptide was identified by electrospray mass spectroscopy to be in the peak eluting at 9% B and was clear of the 10 kDa impurity. The measured mass of 25 093 Da agreed with the value for the calculated mass of the mutant, confirming that the expressed protein was the mutant. The solution was then diluted into folding conditions by adding drop-wise into a tenfold excess of stirred, cooled 25 mM Tris–HCl, pH 7.5, 1 mM DTT. Alternatively, the protein was refolded on a desalting column equilibrated with 20 mM sodium phosphate, pH 7.5, with 1 mM DTT.

A similar procedure for solubilizing the mutant CRYBB2–crystallin was used followed by chromatography under denaturing conditions using anion-exchange chromatography with a HiPrep Q Sepharose FF column at pH 8.0. The unfolded protein eluting at 11–17% buffer 3B was confirmed by electrospray mass spectroscopy to be the mutant as its measured mass of 23 193 Da agreed with the value for the calculated mass of the mutant. The protein was then refolded by drop-wise addition to stirred, cooled 25 mM Tris–HCl, pH 7.5, 1 mM DTT as it was found that considerable protein precipitation occurred if refolding on the desalting column was attempted. The refolded protein was carefully concentrated and then loaded onto a Mono Q column for further purification using the refolding buffer and a salt gradient of refolding buffer with 1 M NaCl.

Preparation of unfolded/refolded wild-type CRYBB2 and wild-type CRYBA3–crystallin
Solutions of the purified wild-type proteins were unfolded by addition of solid urea to make a final concentration of 6 M urea. The unfolded proteins were refolded by adding drop-wise to an excess of a cold, stirred solution of 25 mM Tris–HCl buffer, 1 mM DTT, at pH 8.0 in the case of CRYBB2–crystallin and at pH 7.5, for CRYBA3–crystallin, and equilibrated into native buffer using an Amicon stirred ultrafiltration cell fitted with a YM 30 membrane.

Solubility of the wild-type and mutant crystallins
The wild-type CRYBA3–crystallin, in 20 mM sodium phosphate, pH 7.5, 1 mM DTT could be concentrated by a combination of ultrafiltration and centrifugation in a microconcentrator at 22°C. Protein concentration was estimated by absorption at 280 nm and by the Bradford assay on diluted portions. The solubility of the CRYBA3–crystallin mutant was determined after refolding by dilution into 0.6 M urea, pH 7, 1 mM DTT. The mutant protein could not be concentrated above ~100 µg ml–1. In fact most efforts to handle the protein resulted in its loss. The solubility of the wild-type and mutant ßB2-crystallins were determined using the same procedures.

Secondary structure determination of the proteins
The protein was passed through a desalting column equilibrated with 20 mM sodium phosphate, pH 7.5 for secondary structure estimation using far UV circular dichroism spectroscopy. Spectra were recorded on an AVIV 62DS spectrometer and the data processed using the SUPER3 software (31). Spectra were normalized by the mean residue ellipticity by applying the scale factor (0.1xMRW)/[(mg/ml)xpl] where pl is the cuvette length in centimetres, and MRW is the mean residue weight. Spectra were recorded using a 0.1 cm path length cuvette using protein concentrations:

  • CRYBA3–crystallin wild-type=0.13 mg ml–1;
  • CRYBA3–crystallin wild-type, refolded=0.14 mg ml–1;
  • CRYBA3G91del–crystallin=0.05 mg ml–1;
  • CRYBB2–crystallin wild-type=0.18 mg ml–1;
  • CRYBB2–crystallin wild-type, refolded=0.18 mg ml–1;
  • CRYBB2–crystallin wild-type, refolded=0.21 mg ml–1, and CRYBB2G76del–crystallin, refolded=0.11 mg ml–1.

For measurement of the CRYBA3 wild-type unfolded in 6 M urea, the protein concentration was 1.67 mg ml–1, in a 0.01 cm path length cuvette. The protein concentrations were determined from extinction coefficients derived from the corresponding amino acid sequences as described at: www.iut-arles.up.univ-mrs.fr/w3bb/d_abim/compo-p.html.

The proteins were diluted from a stock solution using 20 mM sodium phosphate buffer pH 7.5. Data were collected over the wavelength range 190–300 nm at 0.2 nm intervals. All spectra were derived from three repeat scans of 20 min. Scans were averaged, smoothed and buffer base line subtracted.

Size estimation of wild-type and mutant CRYBA3 and CRYBB2–crystallins
The protein sample (250 µl) was passed through a Superose 12 10/30 HR column calibrated with low molecular weight markers (Amersham Biosciences) and run at 22°C in 25 mM Tris–HCl, 200 mM NaCl, pH 7.5, at 0.5 ml min–1.


   ACKNOWLEDGEMENTS
 
The authors would like to thank the family for taking part in this study. We would also like to thank Professor Bonnie A. Wallace for many helpful discussions on CD spectroscopy. The work at the Department of Molecular Genetics, Institute of Ophthalmology was supported by a Wellcome Trust grant 063969/Z/01. The work conducted at the Department of Crystallography, Birkbeck College, London was funded by the Medical Research Council.


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +44 12236086800; Fax: +44 2076086863; Email: mareddy{at}doctors.org.uk


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Vijaya, R., Gupta, R., Panda, G., Ravishankar, K. and Kumaramanickavel, G. (1997) Genetic analysis of adult-onset cataract in a city-based ophthalmic hospital. Clin Genet., 52, 427–431.[Web of Science][Medline]

  2. Gilbert, C.E., Canovas, R., Hagan, M., Rao, S. and Foster, A. (1993) Causes of childhood blindness: results from west Africa, south India and Chile. Eye, 7, 184–188.[Medline]

  3. Rahi, J.S. and Dezateux, C. (2000) Congenital and infantile cataract in the United Kingdom: underlying or associated factors. British Congenital Cataract Interest Group. Invest. Ophthal. Visual Sci., 41, 2108–2114.[Abstract/Free Full Text]

  4. Reddy, M.A., Francis, P., Berry, V., Bhattacharya, S.S. and Moore, A.T. (2004) Molecular genetic basis of inherited cataract and associated phenotypes. Surv. Ophthal., in press.

  5. de Jong, W.W., Lubsen, N.H. and Kraft, H.J. (1994) Molecular evolution of the eye lens. Prog. Ret. Eye Res., 13, 391–442.[CrossRef]

  6. Delaye, M. and Tardieu, A. (1983) Short-range order of crystallin proteins accounts for eye lens transparency. Nature, 302, 415–417.[CrossRef][Medline]

  7. Jaenicke, R. and Slingsby, C. (2001) Lens crystallins and their microbial homologs: structure, stability, and function. Crit. Rev. Biochem. Mol. Biol., 36, 435–499.[CrossRef][Web of Science][Medline]

  8. Horwitz, J. (2000) The function of alpha-crystallin in vision. Semin. Cell Dev. Biol., 11, 53–60.[CrossRef][Web of Science][Medline]

  9. Benedek, G.B. (1997) Cataract as a protein condensation disease: the Proctor Lecture. Invest. Ophthal. Visual Sci., 38, 1911–1921.[Free Full Text]

  10. Sandilands, A., Hutcheson, A.M., Long, H.A., Prescott, A.R., Vrensen, G., Loster, J., Klopp, N., Lutz, R.B., Graw, J., Masaki, S. et al. (2002) Altered aggregation properties of mutant gamma-crystallins cause inherited cataract. EMBO J., 21, 6005–6014.[CrossRef][Web of Science][Medline]

  11. Sinha, D., Wyatt, M.K., Sarra, R., Jaworski, C., Slingsby, C., Thaung, C., Pannell, L., Robison, W.G., Favor, J., Lyon, M. and Wistow, G. (2001) A temperature-sensitive mutation of Crygs in the murine Opj cataract. J. Biol. Chem., 276, 9308–9315.[Abstract/Free Full Text]

  12. Kmoch, S., Brynda, J., Asfaw, B., Bezouska, K., Novak, P., Rezacova, P., Ondrova, L., Filipec, M., Sedlacek, J. and Elleder, M. (2000) Link between a novel human gammaD-crystallin allele and a unique cataract phenotype explained by protein crystallography. Hum. Mol. Genet., 9, 1779–1786.[Abstract/Free Full Text]

  13. Pande, A., Pande, J., Asherie, N., Lomakin, A., Ogun, O., King, J. and Benedek, G.B. (2001) Crystal cataracts: human genetic cataract caused by protein crystallization. Proc. Natl Acad. Sci. USA, 98, 6116–6120.[Abstract/Free Full Text]

  14. Francis, P.J., Berry, V., Bhattacharya, S.S. and Moore, A.T. (2000) The genetics of childhood cataract. J. Med. Genet., 37, 481–488.[Abstract/Free Full Text]

  15. Gill, D., Klose, R., Munier, F.L., McFadden, M., Priston, M., Billingsley, G., Ducrey, N., Schorderet, D.F. and Heon, E. (2000) Genetic heterogeneity of the Coppock-like cataract: a mutation in CRYBB2 on chromosome 22q11.2. Invest. Ophthal. Visual Sci., 41, 159–165.[Abstract/Free Full Text]

  16. Litt, M., Carrero-Valenzuela, R., LaMorticella, D.M., Schultz, D.W., Mitchell, T.N., Kramer, P. and Maumenee, I.H. (1997) Autosomal dominant cerulean cataract is associated with a chain termination mutation in the human beta-crystallin gene CRYBB2. Hum. Mol. Genet., 6, 665–668.[Abstract/Free Full Text]

  17. Mackay, D.S., Boskovska, O.B., Knopf, H.L., Lampi, K.J. and Shiels, A. (2002) A nonsense mutation in CRYBB1 associated with autosomal dominant cataract linked to human chromosome 22q. Am. J. Hum. Genet., 71, 1216–1221.[CrossRef][Web of Science][Medline]

  18. Vanita, Sarhadi, V., Reis, A., Jung, M., Singh, D., Sperling, K., Singh, J.R. and Burger, J. (2001) A unique form of autosomal dominant cataract explained by gene conversion between beta-crystallin B2 and its pseudogene. J. Med. Genet., 38, 392–396.[Free Full Text]

  19. Bateman, J.B., Geyer, D.D., Flodman, P., Johannes, M., Sikela, J., Walter, N., Moreira, A.T., Clancy, K. and Spence, M.A. (2000) A new betaA1-crystallin splice junction mutation in autosomal dominant cataract. Invest. Ophthal. Visual Sci., 41, 3278–3285.[Abstract/Free Full Text]

  20. Kannabiran, C., Rogan, P.K., Olmos, L., Basti, S., Rao, G.N., Kaiser-Kupfer, M. and Hejtmancik, J.F. (1998) Autosomal dominant zonular cataract with sutural opacities is associated with a splice mutation in the betaA3/A1-crystallin gene. Mol. Vision, 4, 21.[Medline]

  21. Bateman, J.B., Geyer, D.D., Flodman, P., Johannes, M., Sikela, J., Walter, N., Moreira, A.T., Clancy, K. and Spence, M.A. (2000) A new betaA1-crystallin splice junction mutation in autosomal dominant cataract. Invest. Ophthal. Visual Sci., 41, 3278–3285.[Abstract/Free Full Text]

  22. Kannabiran, C., Wawrousek, E., Sergeev, Y., Rao, G.N., Kaiser-Kupfer, M. and Hejtmancik, J.F. (1999) Mutation of beta-A3/A1 crystallin gene in autosomal dominant zonular cataract with sutural opacities results in a protein with a single globular domain. Invest. Ophthal. Visual Sci. (Abstr.) 40, S786.

  23. Woody, R.W. (1994) Contributions of tryptophan side chains to the far-ultraviolet circular dichroism of proteins. Eur. Biophys. J, 23, 253–262.[CrossRef][Web of Science][Medline]

  24. Bax, B., Lapatto, R., Nalini, V., Driessen, H., Lindley, P.F., Mahadevan, D., Blundell, T.L. and Slingsby, C. (1990) X-ray analysis of betaB2-crystallin and evolution of oligomeric lens proteins. Nature, 347, 776–780.[CrossRef][Medline]

  25. Wieligmann, K., Mayr, E.M. and Jaenicke, R. (1999) Folding and self-assembly of the domains of betaB2-crystallin from rat eye lens. J. Mol. Biol., 286, 989–994.[CrossRef][Web of Science][Medline]

  26. Hemmingsen, J.M., Gernert, K.M., Richardson, J.S. and Richardson, D.C. (1994) The tyrosine corner: a feature of most Greek key beta-barrel proteins. Protein Sci., 3, 1927–1937.[Web of Science][Medline]

  27. Bateman, O.A., Sarra, R., van Genesen, S.T., Kappe, G., Lubsen, N.H. and Slingsby, C. (2003) The stability of human acidic beta-crystallin oligomers and hetero-oligomers. Exp. Eye Res., 77, 409–422.[CrossRef][Web of Science][Medline]

  28. Munier, F.L., Ferrini, W., Othenin-Girard, P., Uffer, S. and Schorderet, D.F. (2003) Autosomal dominant congenital nuclear cataract associated with delG91 in CRYBA3/A1. ARVO Abstract, 5 May.

  29. Lathrop, G.M. and Lalouel, J.M. (1984) Easy calculations of lod scores and genetic risks on small computers. Am. J. Hum. Genet., 36, 460–465.[Web of Science][Medline]

  30. Bateman, O.A., Lubsen, N.H. and Slingsby, C. (2001) Association behaviour of human betaB1-crystallin and its truncated forms. Exp. Eye Res., 73, 321–331.[CrossRef][Web of Science][Medline]

  31. Wallace, B.A. and Teeters, C.L. (1987) Differential adsorption flattening optical effects are significant in the circular dichroism spectra of large membrane fragments. Biochemistry, 26, 65–70.[CrossRef][Medline]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 IOVSHome page
D. Cohen, U. Bar-Yosef, J. Levy, L. Gradstein, N. Belfair, R. Ofir, S. Joshua, T. Lifshitz, R. Carmi, and O. S. Birk
Homozygous CRYBB1 Deletion Mutation Underlies Autosomal Recessive Congenital Cataract
Invest. Ophthalmol. Vis. Sci., May 1, 2007; 48(5): 2208 - 2213.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
H. Liu, X. Du, M. Wang, Q. Huang, L. Ding, H. W. McDonald, J. R. Yates III, B. Beutler, J. Horwitz, and X. Gong
Crystallin {gamma}B-I4F Mutant Protein Binds to {alpha}-Crystallin and Affects Lens Transparency
J. Biol. Chem., July 1, 2005; 280(26): 25071 - 25078.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
13/9/945    most recent
ddh110v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (16)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Reddy, M.A.
Right arrow Articles by Slingsby, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Reddy, M.A.
Right arrow Articles by Slingsby, C.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?