Molecular Vision 2007; 13:2301-2309 <http://www.molvis.org/molvis/v13/a260/> Received 16 October 2007 | Accepted 11 December 2007 | Published 18 December 2007

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Cataract-causing αAG98R mutant shows substrate-dependent chaperone activity

Raju Murugesan, Puttur Santhoshkumar, K. Krishna Sharma
 
 

Departments of Ophthalmology and Biochemistry, University of Missouri, Columbia, MO

Correspondence to: K. Krishna Sharma, Ph.D., Department of Ophthalmology, University of Missouri-Columbia, 1 Hospital Drive, Columbia, MO, 65212; Phone: (573) 882-8478; FAX: (573) 884-4100; email: sharmak@health.missouri.edu

Abstract

Purpose: The G98R mutation in human αA-crystallin is associated with autosomal dominant cataract (presenile type). The reasons for cataract development in αAG98R individuals are not fully understood. Therefore we undertook this study to analyze the stability, structural changes and chaperone function of αAG98R protein.

Methods: Site-directed mutagenesis was employed to generate αAG98R mutant protein. Human αA-crystallin cDNA cloned into the pET23d vector was used as the template. The recombinant proteins were expressed in E. coli and purified using chromatographic methods. Both the wild-type and mutant proteins were characterized by SDS-PAGE, transmission electron microscopy, static and dynamic light scattering, and spectroscopic analysis. The chaperone-like function of the mutant protein was compared with wild-type protein using different substrates.

Results: The G98R mutant protein formed larger oligomers compared to the wild-type αA-crystallin. Circular dichroism studies showed altered secondary and tertiary structure whereas bis-ANS binding studies showed a gain of surface hydrophobicity in the αAG98R protein. The αAG98R protein displayed a substrate-dependent chaperone-like activity. The mutant protein appeared to have diminished chaperone-like activity toward aggregating α-lactalbumin, whereas citrate synthase and alcohol dehydrogenase were efficiently protected from aggregation.

Conclusions: The present results reveal that the G98R mutation causes conformational changes in αA-crystallin and that with certain substrates the mutant protein forms complexes that are prone to precipitate over time. The accumulation of mutant protein-substrate complexes may be the reason for cataract development in individuals carrying the G98R mutation in αA-crystallin.

Introduction

The major lens protein α-crystallin plays an important role in maintaining the transparency of vertebrate eye lens. α-Crystallin exists as polydisperse aggregates with an average molecular mass of 800 kDa [1]. An α-crystallin aggregate consists of two 20 kDa polypeptide chains, αA- and αB-crystallin, that share about 60% sequence homology [2]. The expression of αA-crystallin is reported to be present in the lens, retina, kidney, and thymus, whereas αB-crystallin is reported in brain, spleen, kidney, heart, and skeletal muscle [3,4]. The crystallin subunits belong to members of the small heat shock protein family [5-7]. Like other members of this family, both αA- and αB-crystallin exhibit chaperone-like activity [8,9]. The chaperone function of α-crystallin is essential for maintaining transparency of the lens, which includes preventing the aggregation of unfolding proteins [10-12] and protecting enzymes from inactivation due to heat inactivation [13,14]. Several studies have demonstrated that loss of chaperone activity is associated with cataractous lenses [15-17]. During the aging process, α-crystallin undergoes age-related modification and forms larger aggregates which eventually become water-insoluble proteins [18]. The mechanism of formation of larger aggregates and insolubilization is not known. It may be related to various post-translational modifications such as deamidation [19,20], phosphorylation [21,22], isomerization [23], glycation [15], oxidation [24], truncations [25,26], and disulfide bond formation [27]. These modifications lead to decreased chaperone function and to cataract formation.

In humans, the αA-crystallin gene is located on chromosome 21 and encodes a polypeptide of 173 residues [28]. The αB-crystallin gene is located on chromosome 11 and encodes a 175-residue polypeptide [29]. Several point mutations in αA-crystallin have been reported and these have been linked to cataract in humans. A point mutation at the 413 position in exon 3 alters the highly conserved residue R116 to C in αA-crystallin protein. This mutation results in autosomal dominant congenital cataract [30]. A substitution mutation at 145C>T in exon 1 causes replacement of cysteine for arginine at codon 49 (R49C), which is associated with another autosomal dominant nuclear cataract [31]. Graw et al. [32] reported that a transition mutation at 62C>T in exon 1 leads to substitution of arginine to lysine (R21L), a causative for autosomal dominant cataract. A nonsense mutation at the position 27G>A results in a change of tryptophan to a stop codon W9X [33] and causes autosomal recessive cataract. Similarly, mutations in the αB-crystallin gene have been reported to be associated with cataract. A missense mutation R120G was associated with cataract and desmin-related myopathy [34]. A point mutation D140N [35] and a deletion mutation 450DelA [36] have also been associated with autosomal dominant cataract.

Several laboratories have demonstrated that mutations at arginine residues are associated with loss of positive charge in α-crystallin, causing altered structure and functions. Santhiya and coworkers [37] have reported a new mutation in the αA-crystallin gene (292G>A) which results in substitution of Glycine to Arginine at the 98 position. This mutation leads to a gain of positive charge by αA-crystallin. This genetic mutation was observed in three members of a family in India with onset of cataract at the age of 16 and loss of vision by the age of 24. The molecular mechanism of G98R mutation causing cataract is not fully known. Therefore we undertook this study to analyze the stability and structural changes caused by a mutation at residue 98 in αA-crystallin.

At about the time we began the studies on mutant G98R protein, Singh et al. [16] reported that αA-G98R shows loss of chaperone activity and promotes the DTT-induced aggregation of insulin. During our study, we observed that G98R mutant protein showed significantly more bis-ANS binding than the wild-type αA-crystallin, which indicated that G98R mutant protein has more exposed hydrophobic sites. Since several studies have shown that there is a strong positive correlation between hydrophobicity and chaperone-like activity [38,39] the loss of chaperone-like activity observed in the αAG98R mutant was puzzling [16]. Therefore, we set out to investigate the chaperone activity of αAG98R with different substrates and denaturation methods.

Methods

Human αA-crystallin cDNA (obtained from J.M. Petrash, Washington University, St.Louis, MO) was cloned into the pET-23d (+) vector (Novagen, Madison, WI). This cloned cDNA was used as a template to generate mutation in the αA-crystallin gene using a Quick Change Site Directed Mutagenesis kit (Stratagene, La Jolla, CA) with a set of primers (Forward-5'-GTG GAG ATC CAC AGA AAG CAC AAC GAG-3' and Reverse-5'-CTC GTT GTG CTT TCT GTG GAT CTC CAC-3'). The αAG98R mutation was confirmed by automated DNA sequencing. Both wild-type and mutant proteins were expressed in E. coli BL21(DE3)pLysS cells (Invitrogen, Carlsbad, CA).

Purification of the recombinant wild-type and mutant G98R αA-crystallin

The recombinant proteins were overexpressed using IPTG and the proteins were purified as described earlier [40]. Briefly, bacterial cell pellets were obtained from a one-liter culture, suspended in 10 ml buffer containing 50 mM Tris-HCl (pH 7.2), 100 mM NaCl, and 1 mM EDTA, and sonicated before centrifugation at 17,000 xg for 2 h. Wild-type protein partitioned into the soluble fraction was purified as described earlier [40]. The mutant protein partitioned into the insoluble fraction was washed with Tris-HCl (pH 7.2) buffer and dissolved in the same buffer containing 1 mM EDTA and 6 M urea. The urea-dissolved protein was filtered to remove insoluble aggregates, and the filtrate was directly loaded into an ion-exchange column. The Q-Sepharose column-bound protein was eluted using a stepwise gradient of NaCl (0, 100, 250, and 500 mM) in 50 mM Tris-HCl (pH 7.2) containing 1 mM EDTA at a flow rate of 1 ml/min. The fractions containing recombinant crystallin protein were pooled and concentrated. The purified wild-type αA-crystallin was similarly treated with 6 M urea and separated by ion-exchange chromatography. Wild-type and mutant proteins were analyzed by SDS-PAGE and mass spectrometry to determine their purity. The concentrations of the wild-type and mutant proteins were estimated using Bio-Rad protein assay reagent.

Molecular size determination by multi-angle light scattering

To determine the molecular mass of αA-crystallin, protein samples in 50 mM phosphate buffer containing 150 mM NaCl (pH 7.4) were passed through a TSK5000PW_XL (Tosoh Bioscience, Montgomeryville, PA) gel filtration column attached to a HPLC system, which in turn was linked with a multi-angle laser light-scattering detector, DAWN-EOS (Wyatt Technology, Santa Barbara, CA) and a refractive index detector (Shimadzu, Columbia, MD). Molar mass, hydrodynamic radius (Rh) and polydispersity were estimated as described previously [40].

Tryptophan fluorescence

The intrinsic fluorescence spectra of wild-type and αAG98R proteins were analyzed using a Jasco spectrofluorimeter FP-750 (JASCO Corporation, Tokyo, Japan). Protein samples of 0.2 mg/ml in 50 mM phosphate buffer containing 150 mM NaCl (pH 7.4) were excited at 295 nm and emission spectra were recorded between 300-400 nm.

1,1'-bi(4-anilino) naphthalene-5,5'-disulfonic acid (bis-ANS) fluorescence

The relative surface hydrophobicity of wild-type and αAG98R proteins were measured using bis-ANS. Ten μl of bis-ANS (1 mM) solution was added to 0.2 mg protein in 1 ml buffer consisting of 50 mM phosphate buffer and 150 mM NaCl (pH 7.4). The samples were excited at 385 nm and the emission spectra were recorded between 400- 600 nm using a Jasco spectrofluorimeter FP-750.

Circular dichroism (CD) studies

Changes in protein secondary structure were evaluated by far- and near-UV CD spectra in a JASCO J-815 CD spectrometer (JASCO Inc., Easton, MD). A protein concentration of 0.2 mg/ml was used for far-UV CD measurements, and a concentration of 3 mg/ml was used for near-UV CD measurements. The reported CD spectra are the average of six scans. Secondary structural elements were determined according to a computer software program derived from Sreerama and Woody [41].

Electron microscopic study

The oligomeric structure of the wild-type and αAG98R proteins were observed under a JEOL 1200EX Electron microscope. A drop of protein (1 mg/ml) was applied to a carbon coated grid and negatively stained with 2% uranyl acetate. The specimens were observed at different magnification.

Chaperone-like activity

The following substrates-citrate synthase (CS), α-lactalbumin (α-LA; Sigma, St Louis, MO), and alcohol dehydrogenase (ADH; Biozyme, San Diego, CA) were used to measure the chaperone function of wild-type and mutant αA-crystallin proteins. The extent of aggregation was measured by monitoring the light scattering at 360 nm using a Shimadzu UV-VIS spectrophotometer. CS (75 μg) in 1 ml of 40 mM HEPES-KOH buffer (pH 7.4) was heated at 43 °C in the absence or presence of wild-type or mutant proteins. The ADH aggregation assay was performed at 37 °C by incubating 250 μg of ADH in 50 mM phosphate buffer containing 150 mM NaCl and 100 mM EDTA (pH 7.0) in the absence or presence of wild-type or mutant proteins. For the α-LA aggregation assay, bovine α-LA (0.4 mg) was dissolved in 1 ml of 50 mM sodium phosphate (pH 7.0) containing 100 mM NaCl and 2 mM EDTA, and the aggregation was initiated by the addition of 20 μl of 1 M DTT at 37 °C in the absence or presence of wild-type or mutant proteins.

Thermal stability of wild-type and αAG98R proteins

The wild-type and αAG98R proteins (1 mg/ml), taken in 50 mM phosphate buffer (pH 7.4) containing 150 mM NaCl, were incubated at various temperatures (4 °C, 37 °C, 45 °C, 53 °C, and 65 °C) for 30 min. After incubation the tubes were centrifuged to remove any visible precipitates, and 0.2 ml of the clear supernatant was injected into a TSK G5000PW_XL column. The samples were analyzed on a DAWN-EOS as in the light-scattering studies.

Results

We analyzed the structure, stability and chaperone properties of the recombinant wild-type and αAG98R proteins expressed in E. coli BL21(DE3)pLysS cells and purified by conventional methods. While the wild-type protein was recovered in the soluble fraction following the bacterial cell lysis, the αAG98R protein partitioned into the insoluble fraction, suggesting that mutant protein forms inclusion bodies. The inclusion bodies were dissolved in 6 M urea and purified using Q-Sepharose column chromatography. The mutant protein, once purified, remained soluble enough to permit further analysis. The wild-type αA-crystallin protein was also treated with 6 M urea to maintain identical denaturation-renaturation conditions for both proteins. Figure 1 shows the SDS-PAGE profile of the purified recombinant αAG98R and αA-crystallin. Nanospray QqTOF mass spectrometry of the proteins revealed that the molecular mass was 19,903 Da and 20,002 Da for wild-type and αAG98R, respectively. Electron microscopy studies revealed that both the recombinant wild-type and αAG98R oligomers have an asymmetric and polydisperse appearance. The oligomers of αAG98R were larger and more irregular than the wild-type αA-crystallin oligomers (Figure 2).

Intrinsic tryptophan fluorescence of the proteins measured at 37 °C showed that the emission maximums for both proteins were similar. However, the mutant exhibited 30% higher emission intensity than the wild-type protein (data not shown), indicating a change in the tryptophan microenvironment of the αAG98R protein when compared to wild-type αA-crystallin. bis-ANS is a hydrophobic site-responsive probe which has negligible fluorescence in aqueous media. Binding of this probe to hydrophobic sites in proteins results in a several-fold enhancement in its fluorescence with a shift in the emission maximum to a lower wavelength. The bis-ANS fluorescence emission spectra of αAG98R showed 35% greater intensity than the wild-type αA-crystallin treated with bis-ANS, suggesting increased exposure of hydrophobic region(s) (data not shown).

Secondary structural analysis of wild-type and αAG98R was performed by scanning the proteins at the far-UV range, 240 nm to 198 nm. The amide backbone of the protein absorbs in the far ultraviolet region and produces signals which enable us to determine the secondary structural details of the protein. The far-UV CD spectra of wild-type αA-crystallin exhibit a maximum negative ellipticity at 218 nm, indicating that β-sheet content is greater in wild-type protein. On the other hand, the αAG98R protein showed increased negative ellipticity at 208 nm, indicating that the mutant protein has increased α-helical content. The secondary structural elements estimated showed a marginal increase in α-helix content in the G98R mutant protein compared to the wild-type αA-crystallin (4.7% and 2.1%, respectively). In addition, a marginal decrease was observed in β-sheet (38.9%) and random coil (33.8%) content in αAG98R compared to wild-type αA-crystallin with 41.0% (β-sheet) and 34.1% random coil content (Figure 3A).

The near-UV CD spectrum of G98R mutant protein was similar to wild-type αA-crystallin in the 270 nm to 250 nm region. Both proteins exhibit the maxima at 259 nm and 265 nm that is the characteristic feature of phenylalanine fine structure. However, the spectrum in the 300 to 270 nm region was significantly altered in the αAG98R protein compared to the wild-type αA-crystallin. The fine structure of this region is contributed by tyrosine and tryptophan residues. It is evident from Figure 3B that the packing of the aromatic residues Tyr and Trp was significantly altered in the mutant protein.

We have investigated the molecular mass of the wild-type and mutant protein by multi-angle light-scattering with online gel filtration chromatography. The average oligomeric mass calculated from light-scattering studies was 670 kDa for wild-type αA-crystallin with a polydispersity of 1.053. Under similar conditions, the αAG98R protein had an average oligomeric mass of 3,400 kDa with a polydispersity of 1.157. The theoretically estimated number of subunits per oligomer of wild-type αA-crystallin was 33. It is significantly increased to 170 subunits per oligomer of G98R mutant αA-crystallin. The Rh of the wild-type αA-crystallin was 8.5 nm, and the Rh of the G98R mutant proteins was 16 nm, indicating a larger oligomeric size. During gel filtration chromatography the mutant protein elutes earlier than the wild-type protein, once again confirming the larger oligomeric size of the G98R mutant protein (Figure 4 and Table 1).

We examined the chaperone-like property of the wild-type and αAG98R proteins using 3 different substrates: ADH, CS, and αLA. The G98R mutant protein exhibited better chaperone activity than the wild-type protein in CS (Figure 5A) and ADH (Figure 5B) aggregation assays. However, when DTT-induced αLA aggregation assays were used to compare the chaperone activity of wild-type and αAG98R protein, the results were surprising. G98R mutant protein prevented the aggregation of αLA for up to 50 min of the reaction time (Figure 5C). Then the substrate and the G98R-crystallin complex (confirmed by SDS-PAGE analysis of the aggregate, data not shown) began to form light-scattering aggregates.

To check the stability of the proteins, wild-type αA-crystallin and αAG98R were incubated at various temperatures (4 °C, 37 °C, 45 °C, 53 °C, and 65 °C) and analyzed by size exclusion chromatography and light-scattering measurements. Wild-type protein remained clear and stable in solution up to 53 °C (Figure 6). However, the mutant protein formed light-scattering aggregates at and above 45 °C. This suggests that the mutant protein is sensitive to temperatures slightly above the normal physiologic condition. The wild-type protein formed large aggregates only at 65 °C. The average oligomeric mass of wild-type protein was 694 kDa at 4 °C, which decreased to 568 kDa on increasing the incubation temperature to 37 °C (Table 1). Increasing the temperature beyond 37 °C slightly enhanced the oligomeric mass of wild-type αA-crystallin. The wild-type protein remaining in solution after 65 °C treatment had an average mass of 19.5 mDa. In contrast the αAG98R had an average oligomeric mass of 2.65 mDa at 4 °C, which increased to 7.08 mDa at 37 °C. At 45 °C part of the αAG98R precipitated and the oligomers that remained in solution had aggregate sizes ranging from 12 - 438 mDa. The Rh of the protein incubated at different temperatures is shown in Table 1. The Rh of wild-type αA-crystallin decreased from 8.5 nm to 7.9 nm when the incubation temperature was increased from 4 °C to 37 °C. Increasing the incubation temperature beyond 37 °C increased the Rh. At 65 °C the Rh of the wild-type protein that remained in the soluble form was 25.8 nm. In contrast, the Rh of the αAG98R was 15.2 nm at 4 °C, which increased with increasing incubation temperature. At 45 °C the Rh of the αAG98R remaining in solution was 30.2 nm. It was not possible to analyze the mass of the αAG98R beyond this temperature due to precipitation of proteins.

Discussion

The novel αA-crystallin mutation G98R was identified and reported in an Indian family by Santhiya et al. [37]. G98R is the only mutation in αA-crystallin associated with cataract where a positively charged amino acid replaces the uncharged amino acid in the native protein. The other known mutations in αA-crystallin, such as R116C, R41C, and R21L have a substitution of a uncharged amino acid [30-32]. Therefore, we investigated the effect of the G98R mutation on the structure and function of αA-crystallin. Using a site-directed mutagenesis method, we constructed the αAG98R mutant and expressed it in E. coli cells. The mutant αA-crystallin protein was expressed but the recombinant protein formed inclusion bodies. The protein from the inclusion bodies was solubilized with urea and refolded. Interestingly, the urea-refolded mutant proteins stayed in soluble form. We observed that when the recombinant protein was extracted within 2 h of IPTG induction G98R was present in both water-soluble and water-insoluble fractions. This suggests that at higher concentrations the mutant proteins is more prone to aggregation and forms inclusive body in bacterial cells.

The present study suggests that mutant protein is sensitive to temperature. When mutant αAG98R and wild-type αA-crystallin proteins were incubated at various temperatures (4 °C, 25 °C, 37 °C, 45 °C, 53 °C, and 65 °C) the wild-type protein was found to be stable at 53 °C and large aggregates of wild-type αA-crystallin were observed at 65 °C. The aggregation of wild-type αA-crystallin is similar to the earlier report that recombinant αA-crystallin shows a transition between 55 °C and 65 °C and undergoes structural alteration [42]. Unlike wild-type αA-crystallin, the mutant G98R protein formed larger aggregates as the temperature of the sample was raised from 37 °C to 45 °C. The hydrodynamic radius and the polydispersity of the aggregate also increased (Table 1). This data suggests that the mutant protein is prone to aggregation at near-physiologic temperature.

Figure 5A shows that the mutant protein prevents heat-induced aggregation of CS. Interestingly, G98R mutant protein showed higher chaperoning activity than the wild-type αA-crystallin at the same molar ratio when CS is used as substrate. The increased chaperone-like activity correlates with the increased hydrophobicity reflected by enhanced bis-ANS binding to the G98R mutant. Several earlier studies have shown that there is a correlation between increased hydrophobicity and chaperone activity of αA-crystallin [38,43-45]. It is yet to be determined whether the increased chaperone activity is due to the accessibility of new chaperone sites on mutant protein or due to the enhanced affinity of G98R to denaturing substrates. The G98R mutant protein, however, showed a decreased chaperone activity when aggregation assays were performed using DTT and αLA. Although the mutant protein suppressed DTT-induced aggregation of αLA for the initial 40 min, continuation of the assay resulted in precipitation of the protein-substrate complex. Recently, Singh et al. [16,46] reported that G98R mutant protein failed to protect DTT-induced aggregation of Insulin. The authors also suggested that the mutant protein has the ability to interact with target protein, and the protein-substrate complex become water-insoluble. The molecular mechanism of precipitation of G98R mutant protein along with substrate is not known. But it appears that the interaction of G98R with insulin [16,46] or α-LA (this study) is similar to the interaction of R116C and R49C with substrate proteins where the mutant crystallin showed increased affinity toward the substrate proteins, interpreted as "gain of function" or "activation of the chaperone site" [47]. It is likely that as in the case of R116C [47], when the chaperone sites in mutant proteins become saturated with the denaturing αLA, the ability of the mutant oligomers to keep the complex in soluble form diminishes. Additionally, it may be possible that the αLA undergoes conformational changes upon binding to the mutant crystallin as shown previously [48] and the resulting complex has diminished solubility. Previous study with mini-chaperone [49] has also indicated that it has different activity toward different substrate proteins. The different degrees of chaperone activity on different substrates indicates that αA-crystallin may have more than one target protein binding site, and that one of the binding sites is affected by the mutation while the unaffected site continues to function as a chaperone site. Further studies are required to confirm this. We have reported earlier that 89VLGDVIEVHGK99 is one of the substrate binding sites in αA-crystallin [45] and that interestingly, the G98R mutation occurs at this binding site.

Earlier studies reported that α-crystallin exhibits better chaperone function at slightly elevated temperatures when compared to the activity at 37 °C [42,47,50]. In the present study we observed that the mutant G98R protein exhibits decreased chaperone activity at 43 °C (Figure 7) compared to the activity at 37 °C (Figure 5B). Since we have also observed that at slightly elevated temperatures the mutant protein tends to aggregate, it is likely that the mutant forms aggregate with decreased chaperone activity at higher temperatures. Additionally, the complex formed between mutant and substrate may become less stable due to the decreased stability of one of the partners in the complex (G98R).

Individuals who carry the G98R mutations start developing cataract when they are in their late teens. It has been reported that one individual with mutant G98R αA-crystallin had clear vision up to the age of 16 then decreased vision followed by complete loss of vision and mature cataract by the age of 24. Since most of the mutations result in congenital cataract the reasons for the time lag is not clear. It is possible that due to the presence of wild-type αB-crystallin, αAG98R functions efficiently in early life but when the patients are in their teens the insult to lens proteins overwhelms the capacity of the functional chaperone protein and the proteins start precipitating, with ensuing cataract. To test this we performed a chaperone function assay with different ratios of mutant G98R and wild-type αA- or αB-crystallin. The results of one such experiment is shown in Figure 8. The severity of aggregation is proportional to the percentage of mutant proteins in the mixture. This data suggests that the delayed onset of cataracts may be due to the presence of wild-type αB-crystallin in vivo and the age-related post-translation modifications [18,21,22] may be diminishing the chaperone function of αB-crystallin, thereby limiting its ability to protect the lens from developing an opacity. Further, due to its decreased stability the G98R protein may also be aggregating in vivo and contributing to the development of cataract.

In conclusion, the present study confirms the finding of an earlier study [16] which reported that the G98R mutation in αA-crystallin significantly alters the secondary and tertiary structure of the protein and the mutant is highly sensitive to temperature and shows decreased chaperone activity during insulin reduction assay. In addition, during our investigation we observed that G98R forms irregular shape oligomers larger than that of wild-type protein and exhibits differential chaperone activity. The G98R, unlike R116C mutant of αA-crystallin [17,51] showed enhanced chaperone activity when CS or ADH was used as substrate. Further, the chaperone assays involving different ratios of αB-crystallin and G98R performed during the current study demonstrate that the age-onset cataract in individuals carrying the G98R mutation may be due to the saturation of in vivo chaperone activity of αB-crystallin. Since the individuals with G98R develop cataract during teen age period or there after [37], it can be argued that the enhanced chaperone activity of G98R observed during an in vitro assay with CS or ADH may not be translating into long-term persisting in vivo chaperone activity. Instead, it is likely that the "gain of function" hypotheses eluded earlier [51] and the propensity of G98R-substrate complex to precipitate in vivo results in lens opacity in patients carrying the mutation.

Acknowledgements

This work was supported by National Institutes of Health Grants EY 11981 and EY14795 and a grant-in-aid from Research to Prevent Blindness.

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