αA-Crystallin–Derived Mini-Chaperone Modulates Stability and Function of Cataract Causing αAG98R-Crystallin

Background A substitution mutation in human αA-crystallin (αAG98R) is associated with autosomal dominant cataract. The recombinant mutant αAG98R protein exhibits altered structure, substrate-dependent chaperone activity, impaired oligomer stability and aggregation on prolonged incubation at 37°C. Our previous studies have shown that αA-crystallin–derived mini-chaperone (DFVIFLDVKHFSPEDLTVK) functions like a molecular chaperone by suppressing the aggregation of denaturing proteins. The present study was undertaken to determine the effect of αA-crystallin–derived mini-chaperone on the stability and chaperone activity of αAG98R-crystallin. Methodology/Principal Findings Recombinant αAG98R was incubated in presence and absence of mini-chaperone and analyzed by chromatographic and spectrometric methods. Transmission electron microscope was used to examine the effect of mini-chaperone on the aggregation propensity of mutant protein. Mini-chaperone containing photoactive benzoylphenylalanine was used to confirm the interaction of mini-chaperone with αAG98R. The rescuing of chaperone activity in mutantα-crystallin (αAG98R) by mini-chaperone was confirmed by chaperone assays. We found that the addition of the mini-chaperone during incubation of αAG98R protected the mutant crystallin from forming larger aggregates that precipitate with time. The mini-chaperone-stabilized αAG98R displayed chaperone activity comparable to that of wild-type αA-crystallin. The complexes formed between mini-αA–αAG98R complex and ADH were more stable than the complexes formed between αAG98R and ADH. Western-blotting and mass spectrometry confirmed the binding of mini-chaperone to mutant crystallin. Conclusion/Significance These results demonstrate that mini-chaperone stabilizes the mutant αA-crystallin and modulates the chaperone activity of αAG98R. These findings aid in our understanding of how to design peptide chaperones that can be used to stabilize mutant αA-crystallins and preserve the chaperone function.


Introduction
Alpha A-crystallin, a major structural protein of the vertebrate eye lens [1], belongs to the small heat shock protein (Hsp) family [2][3][4]. Like other members of this family, aA-crystallin exhibits chaperone-like activity [5][6][7][8][9]. The chaperone function of aAcrystallin prevents aggregation of unfolding proteins and is essential for maintaining transparency of the lens [6,7]. In humans, the aA-crystallin gene is located on chromosome 21 and encodes a polypeptide of 173 residues [10]. Several point mutations have been reported in human aA-crystallin and these mutations cause structural changes in the protein and impair its chaperone activity. The loss of chaperone activity is considered one of the causes for the development of cataract [7,[11][12][13]. Congenital cataract is associated with R12C [14], R21L [15], R49C [16], R54C [17], and R116C [18] mutations, which occur at the conserved arginine residues. Pre-senile cataract is associated with a novel G98R mutation in aA-crystallin [19]. In the G98R mutation, a bulky basic amino acid, arginine, replaces the neutral glycine. Earlier studies of the recombinant G98R mutant protein revealed altered structure, substrate-dependent chaperone activity and impaired oligomer stability compared to wild-type recombinant aA-crystallin [20][21][22] Generally, mutant proteins are prone to misfolding in the endoplasmic reticulum (ER) and subsequent aggregation. Paradoxically, some mutant proteins seem to fold efficiently in the ER but are subsequently misfolded at their target sites due to modification in their microenvironment. Functionally impaired mutant proteins or protein aggregates are generally rapidly degraded by the intracellular quality-control system [23] but some escape the quality-control mechanisms. Misfolded proteins are a hallmark of several pathological conditions including cataract. Several lines of evidence suggest that small molecular chaperones would be potential therapeutic molecules for diseases associated with misfolded proteins. Collectively called pharmacological chaperones, such molecules include native ligands, substrate analogues and small peptides [24,25], which bind to mutant proteins and stabilize the mutant proteins to the extent that they function normally in vivo as well as in vitro.
We identified the major chaperone site in aA-crystallin and demonstrated that a 19 amino acid peptide (aA70-88, KFVIFLDVKHFSPEDLTVK), representing the chaperone site in the protein, functions like a molecular chaperone [26]. We have designated such a peptide as a ''aA-mini-chaperone.'' The amino acid sequence of this mini-chaperone is a highly conserved region among several small Hsps [27] and structure analysis shows that aA-mini-chaperone region aligns to the b3 and b4 region in the aA-crystallin. Our studies revealed that the aaA-mini-chaperone is effective in suppressing aggregation of H 2 O 2 -induced xcrystallin [28] and denaturing substrate proteins ADH, citrate synthase, insulin and a-lactalbumin [26,29,30]. The mini-chaperone also inhibits amyloid fibril formation and its toxicity [31]. Because both b-sheet structure and hydrophobicity are necessary for maximal activity of the mini-chaperone, we concluded that direct interaction between the chaperone peptide and client protein is responsible for chaperone-like activity.
In this study, we examined the effect of aA-crystallin-derived mini-chaperone on the stability and function of the mutant aAcrystallin G98R. We show that mini-chaperone stabilizes the unstable mutant protein. Compared to the mutant protein, the mini-chaperone-stabilized aAG98R has a better capacity to chaperone denaturing protein. Our studies demonstrate specific interaction between the mini-chaperone and the mutant aAcrystallin. Using synthetic mini-chaperone harboring a benzoyl phenylalanine (Bpa) residue in place of a Phe we found that the mini-chaperone interacts at least at 1:1 ratio with mutant aAG98R subunits and the stabilized protein has the chaperone activity comparable to that of the WT-aA-crystallin.

Proteins and peptides
Recombinant wild-type aA-crystallin and aAG98R mutants were expressed and purified as described earlier [21]. In brief, the full-length human aA-crystallin cDNA cloned into pET-23d (+) vector (Novagen, Madison, WI) was used as a template to generate the G98R mutation. Both mutant and wild-type proteins were expressed in E. coli BL21(DE3)pLysS cells (Invitrogen, Carlsbad, CA) and purified by column chromatography. The purity of the proteins was checked by SDS-PAGE and the molecular mass was determined by mass spectrometry. The concentration of the mutant and wild-type protein was estimated using Bio-Rad protein assay reagent. Mini-chaperone peptide (DFVIFLDVKHF-SPEDLTVK), also called aA-mini-chaperone, and Pro-substituted mini-chaperone (DFVPFLDVKHFSPEDLTVK) were supplied by GenScript Corp. (Piscataway, NJ). Biotin-DFVIFLDVKH(Bpa)SPEDLTVK was supplied by Aapptec (Louisville, KY). The peptides used in the study were .95% pure as determined by high-performance liquid chromatography (HPLC) and mass spectrometry (MS). Alcohol dehydrogenase (ADH) was obtained from Biozyme, (San Diego, CA). All other chemicals were of the highest grade commercially available.
Aggregation and multi-angle light scattering studies aAG98R or wild-type aA-crystallin (75 mg) were incubated in the presence and absence of aA-crystallin-derived mini-chaperone (10 mg) for 1 hr in 100 ml PO4 buffer at 43uC, the temperature at which aAG98R readily aggregates [21]. Samples were injected on to a TSK G5000PW XL (Tosoh Bioscience, Montgomeryville, PA) size-exclusion column equilibrated with 50 mM sodium phosphate buffer containing 150 mM NaCl (pH 7.2). The flow rate was set to 0.75 ml/min. The column was attached to a HPLC system connected with UV and refractive index detectors and coupled to a static multi-angle laser light scattering (DAWN-EOS) and dynamic quasi-elastic light scattering detectors (Wyatt Technology, Santa Barbara, CA). The molar mass (Mw), hydrodynamic radius (Rh) and polydispersity index (PDI) were determined using ASTRA (5.3.2) software developed by Wyatt Technology.

Fluorescence spectroscopy
For measurement of intrinsic Trp fluorescence, protein samples (200 mg) were diluted in 1 ml of PO4 buffer (50 mM, pH 7.2, containing 150 mM NaCl) in the absence and the presence of mini-chaperone (10 mg). The sample was excited at 295 nm (slit width 5 nm) and the emission was recorded at 300-400 nm range (slit width 5 nm). The relative surface hydrophobicity of wild-type aA-crystallin and aAG98R proteins was measured using bis-ANS. Bis-ANS (1 mM) solution, 10 ml, was added to 0.2 mg protein in 1 ml buffer (50 mM phosphate buffer containing 150 mM NaCl, pH 7.2) in the absence and in the presence of mini-chaperone (10 mg). The samples were excited at 385 nm and the emission spectra were recorded between 400-600 nm using a Jasco spectrofluorimeter FP-750.
Effect of aA-crystallin-derived mini-chaperone on chaperone activity measurements The chaperone-like activity of wild-type aA-crystallin and aAG98R proteins was determined in the presence and absence of mini-chaperone using denaturing ADH as the aggregating substrate. Aggregation assay was performed in 1 ml 50 mM phosphate buffer containing 150 mM NaCl and 100 mM EDTA at 43uC. The chaperone activity of Biotin-DFVIFLDVKH(Bpa)S-PEDLTVK as well as a Pro-substituted mini-chaperone (DFVPFLDVKHFSPEDLTVK) was also measured by ADH aggregation assay. The extent of aggregation was estimated by monitoring the light scattering at 360 nm using a Shimadzu UV-VIS spectrophotometer equipped with a temperature-controlled multi-cell transporter.

Circular dichroism measurements
Circular dichroism (CD) spectropolarimeter, J815 (Jasco, Easton, MD), equipped with a temperature control system, was employed to record CD spectra. Far-UV CD measurements were carried out over the wavelength range of 190 to 250 nm with bandwidth 0.5 nm, scan speed 10 nm/min using 0.1-cm path length cuvettes. Protein samples were prepared in 10 mM sodium phosphate buffer (pH 7.2). Spectra are the average of five scans. Buffer signal was subtracted prior to reporting the data. Thermal denaturation data were collected from 25uC to 45uC, with protein concentration of 100 mg/400 ml. Thermal denaturation experiments were performed with a heating rate of 1uC/min, and CD signals at 218 nm were used to determine transition midpoints. Near-UV CD spectra were recorded using a protein sample of 1.5 mg/ml in the buffer used for far-UV studies.
Confirmation of aA-crystallin-derived mini-chaperone binding to aAG98R protein The interaction between aA-crystallin-derived mini-chaperone and aAG98R-crystallin was studied using benzoyl phenylalaninesubstituted aA-mini-chaperone. Phenylalanine corresponding to Phe-80 in wild-type aA-crystallin was substituted in aA-minichaperone with benzoyl-phenylalanine (Bpa). aA-Mini-chaperone also contained a biotin moiety at the N-terminus, creating a biotinyl aA-mini-chaperone-Bpa peptide. Biotinyl-aA-mini-chaperone-Bpa peptide (200 mg) was incubated with 200 mg of aAG98R crystallin in buffer, pH 7.2, at 37uC for 1 hr. Subsequently, the sample was filtered in a Microcon 10 kDa cut of filter (Millipore, Bedford, MA) to remove free peptides. The sample was photolyzed for 30 min, at 4uC, using a UV lamp (365 nm) held at a distance of 7 cm from the sample. The photolyzed sample was desalted using C18 zip tip spin columns (Thermo Fisher Scientific, Rockford, IL), as per the manufacturer's protocol, and the bound protein was eluted in 70% acetonitrile. The binding of aA-minichaperone to aAG98R was confirmed by MALDI-TOF/TOF mass spectrometry. The UV-photolyzed sample was subjected to SDS-PAGE and western blot analysis using antibody against biotin.

Electron microscopy
To examine the aggregation of G98R mutant protein (100 mg) in the absence and presence of aA-crystallin-derived minichaperone (10 mg), the purified protein was incubated at 37uC or 40uC in 7.2 pH phosphate buffer and the samples were analyzed by transmission electron microscopy (TEM). Aliquots of 5 ml were withdrawn at different time intervals (0 min, 10 min, 30 min) and placed on carbon-coated, 200 mesh copper grids and left for 1 min. The excess solution was wicked away with a filter paper. The proteins on the grid were stained with 5 ml of freshly prepared 5% uranyl acetate solution for 10 min. This solution was then wicked off, and the grid was air-dried and then examined using a JEOL 1400 TEM (120 kV). The images were captured on a digital camera with 20,000 magnification and imaging software from Gatan Digital Micrograph (Gatan, Inc., Warrendale, PA). The protein samples incubated at 37uC in presence and absence of mini-chaperone for 8 hrs and processed as above was also examined by TEM.

Results
Recombinant crystallin proteins were expressed and isolated according to the procedure described earlier [21]. SDS-PAGE analysis confirmed that both wild-type and mutant forms of recombinant aA-crystallins were as pure (.98%) as the proteins used in earlier studies [21,22]. Size-exclusion chromatographic profile of the mutant protein gave an elution profile with an oligomer peak and a peak of dissociated subunits, indicating that the mutant protein has an unstable oligomeric assembly, as described earlier [22]. On the other hand, the wild-type aAcrystallin eluted from the same column as a single peak with the expected elution time for aA-crystallin oligomer. Incubation of aAG98R at 37uC led to gradual aggregation over a period of time, whereas incubation at 43uC resulted in rapid aggregation of the mutant protein, as we reported earlier [21].
aA-Crystallin-derived mini-chaperone increases the recovery of soluble aAG98R Following purification ofaAG98R, we examined the ability of aA-mini-chaperone to stabilize the mutant protein. We know from previous studies thataA-mini-chaperone suppresses the aggrega-tion and precipitation of denaturing proteins [26,29,30]. The purified aAG98R (75 mg), which aggregates and precipitates on incubation at 37-45uC [21] was incubated in the presence and absence of aA-mini-chaperone (10 mg) at 43uC for 1 hr. The samples were centrifuged to remove any precipitate formed during incubation, and the supernatant was analyzed by TSKG5000 PW XL gel filtration column connected to a multi-angle laser light scattering (DAWN-EOS) and dynamic quasi-elastic light scattering detectors. The elution profile showed two peaks ( Figure 1B). The first peak corresponded to the oligomeric form of aAG98R, whereas the second peak represented dissociated subunits of the mutant protein. In the absence of mini-chaperone, only 7.1 mg (9.5%) of the mutant protein was recovered, whereas 59.2 mg (79%) of the mutant protein was recovered when the incubation was carried out with aA-mini-chaperone. The monomeric peak also decreased in the presence of aA-mini-chaperone. The binding of aA-mini-chaperone to aAG98R was confirmed by HPLC analysis of the protein peak eluting between 8.5-11 min from the TSKG5000PW XL column (the data are shown in Figure S1). Both aAG98R and aA-mini-chaperone were present in the protein peak, indicating that the peptide chaperone was in complex with aAG98R during gel filtration analysis. The average molar mass ofaAG98R oligomer (non-aggregated) recovered in the absence of aA-crystallin-derived mini-chaperone was 2.3610 6 , whereas in the presence of the mini-chaperone, the average molar mass of the stabilized aAG98R was 2.8610 6 , indicating that the aA-minichaperone prevented the dissociation of aAG98R protein and that the slightly increased molar mass might be due to binding of aAmini-chaperone ( Figure 1B). The hydrodynamic radius (Rh) of the stabilized aAG98R increased from 15.3 nm to 16.4 nm, consistent with increase in molar mass. Under similar experimental conditions, wild-type aA-crystallin oligomeric size and molar mass did not change upon incubation at 43uC ( Figure 1A) and the minichaperone did not interact with wild-type aA-crystallin. This was confirmed by HPLC analysis of wild-type protein oligomer incubated with aA-mini-chaperone and isolated by gel filtration ( Figure S1).

Stabilization of recombinant aAG98R by aA-crystallinderived mini-chaperone
To investigate the thermal behavior of mutant aAG98R protein and the effect of aA-mini-chaperone on aAG98R stability, we incubated the mutant protein (750 mg) at 43uC in the presence and absence of mini-chaperone, in a 1.7:1 (mol/mol) ratio. Light scattering was monitored at 360 nm for 90 min using a spectrophotometer. As shown in Figure 2, aAG98R begins to form light scattering aggregates in 40 min. It is well known that under similar conditions, the wild-type aA-crystallin does not form light scattering aggregates. The chaperone peptide DFVIFLDVKHFSPEDLTVK, is known to suppress aggregation of proteins denatured by heat [26], chemicals [30] and oxidation [28], completely suppressed aAG98R aggregation ( Figure 2). Because aAG98R is a structurally perturbed protein [20,21], we hypothesize that the mini-chaperone interacted with mutant aAG98R and prevented aggregation and light scattering. Under similar conditions, incubation of aAG98R with a Pro substituted mini-chaperone (DFVPFLDVKHFSPEDLTVK), which has no chaperone activity ( Figure S2), failed to suppress precipitation of the mutant protein (data not shown). The aggregation and precipitation of aAG98R also occurred at 37uC but at a slower rate. It took ,8 hrs to see light scattering by aAG98R at 37uC and addition of aA-mini-chaperone completely suppresses light scattering (data not shown). In a separate experiment, when different amounts (1-30 mM) of mini-aA-crystallin was used with 10 mM of aAG98R in incubations at 43uC for 30 min, there was an increased recovery of mutant protein in soluble form from the reaction mixtures that contained higher amounts of chaperone peptide. Nearly 80% of aAG98R was recovered when the incubation was carried out with 1:2 ratio (mol/mol) of aAG98R to mini-aA-. Further analysis of the aAG98R recovery data gave a Kd value 5.1 mM indicating the peptides high affinity to mutant protein.

Morphology of aAG98R aggregates and stabilization by aA-crystallin-derived mini-chaperone
We examined under TEM the aAG98R incubated at 40uC to observe the aggregation pattern over a 30 min period. During TEM visualization the aAG98R incubated at 40uC showed formation of smaller aggregates comprising 2-10 oligomers in 10 min ( Figure 3D). With longer incubation, several oligomers came together to form larger aggregates, and after 10 min of incubation, the smaller aggregates coalesced to give an amorphous appearance in 30 min ( Figure 3E). Further we also observed that the aAG98R oligomer size of ,15 nm gradually increased to a larger asymmetric form (,20 nm) in ,10 minutes of incubation at 40uC. Further incubation of the mutant protein resulted in larger, irregularly shaped aggregates that precipitate. However, aAG98R incubated in the presence of aA-mini-chaperone, in a mutant-to-peptide chaperone ratio of 1: 0.9 (mol/mol), did not form clusters or amorphous aggregates of oligomers (Figure 3 F). Additionally, the size of aAG98R oligomer incubated with aAmini-chaperone was slightly smaller than the mutant incubated alone for 10 min at 40uC. A similar pattern of aggregation and suppression of aggregation with mini-chaperone was also observed by TEM when the mutant protein was incubated at 37uC for 8 hrs (compare B and C in Figure 3).
Structural changes in aAG98R in the presence of aAcrystallin-derived mini-chaperone The thermal behavior of aAG98R mutant in the presence and absence of aA-mini-chaperone was investigated at both near-UV and far-UV range, using a CD spectrometer equipped with a temperature controller. The temperatures of wild-type aAcrystallin and aAG98R mutant samples, from 25uC to 45uC, were raised slowly and negative ellipticity was recorded. The far-UV CD-spectra showed that wild-type aA-crystallin is very stable until 40uC, and at temperature above 40uC, the negative ellipticity at 218 nm increased with increasing sample temperature, indicating structural changes in the protein ( Figure 4A). The mutant aAcrystallin began to show a significant increase in ellipticity above 27uC, and at temperatures above 40uC, the increases in ellipticity were moderated. Incubation aAG98R with aA-crystallin minichaperone stabilized the protein, as the negative ellipticity at 218 nm remained stable up until 35uC. Above 35uC, there was a gradual increase in 218 nm ellipticity, suggesting structural changes in the mutant protein occur at these temperatures even in presence of mini-chaperone. The near-UV CD spectrum of aAG98R-mini-chaperone was similar to that of wild-type protein in 272-260 nm region, whereas the spectrum in the 300-272 nm region showed minor changes suggestive of interactions between aAG98R and the mini-chaperone ( Figure 4B) but the minimal nature of the interaction may be indicative of the interactions occurring away from the aromatic residues. This is supported by the absence of the peptide effect on intrinsic tryptophan fluorescence of aAG98R ( Figure S3).  Chaperone activity of aAG98R is stabilized after interaction with aA-crystallin-derived mini-chaperone Earlier we reported that, compared to wild-type aA-crystallin, aAG98R mutant protein showed chaperone activity against denaturing ADH at 37uC during the early phase of the assay [21]. However, aAG98R chaperone activity diminished after 30 min of reaction at 43uC and precipitation of proteins was observed [21]. SDS-PAGE analysis of the precipitate revealed that aAG98R protein co-precipitated along with substrate ADH. We postulated that the precipitation was due to the unstable nature of ADH-aAG98R complex, and investigated whether aA-minichaperone would stabilize the complex. Similar to our earlier observation [21], aAG98R suppressed ADH aggregation during the early part of the 2 hr assay but the assay mixture started to scatter light after 60 min ( Figure 5A). However, the addition of aA-crystallin-derived mini-chaperone to the reaction mixture of ADH+aAG98R significantly reduced the aggregation of denaturing protein ( Figure 5A). It should be noted that the addition of aAmini-chaperone did not solubilize the aggregates already formed. The suppression of aAG98R aggregation beyond the point of the addition of aA-mini-chaperone could be either due to the effect of aA-mini-chaperone itself or due to the stabilization of the ADH-aAG98R complex by the aA-mini-chaperone. To examine the latter possibility, aAG98R protein was incubated with aA-minichaperone at 37uC for 30 min, and the aAG98R-aA-minichaperone complex was isolated by gel filtration using a TSK G5000PW XL column and the chaperone activity of the complex was determined employing ADH aggregation assay. The aAG98R treated with aA-mini-chaperone exhibited better chaperone activity than the untreated aAG98R ( Figure 5B). Further, the chaperone activity of aA-mini-chaperone-stabilized mutant protein was comparable to that of wild-type aA-crystallin.

Confirmation of aA-crystallin-derived mini-chaperone binding to aAG98R
We took advantage of photoaffinity labeling with Bpa, which was incorporated into the aA-mini-chaperone at one of the Phe sites, to elucidate the aA-mini-chaperone interaction with aAG98R. The biotin at the N-terminal of the peptide chaperone allowed the detection of the aA-mini-chaperone-G98R complex. Since biotin was attached at the N-terminus and the Bpa group was away from the critical Phe (corresponding to Phe 71 in aAcrystallin), the aA-mini-chaperone retained chaperone activity after these modifications. The photoaffinity labeling of aAG98R was performed using biotin-labeled Bpa-mini-aA and aAG98R. Excess Bpa-mini-chaperone was removed by filtration prior to photolysis. The photolyzed protein was analyzed by SDS-PAGE and western blot using avidin-horseradish peroxidase conjugate against biotin and mass spectrometry. Western blot of UVirradiated mixture of aAG98R and Bpa-mini-aA separated by SDS-PAGE showed the presence of aAG98R-Bpa-mini-aA crosslinked protein band (Figures 6). The molecular weight of the biotin-containing protein band suggests that one peptide was incorporated into one subunit of aAG98R during photolysis. Image analysis of the stained gel showed that mini-chaperone-aAG98R had photo-crosslinked about 10% of aAG98R. MALDI TOF/TOF mass spectrometric profile of the photolyzed sample also showed that about 10% of aAG98R was bound with one biotin-Bpa-peptide ( Figure 7B), whereas the unphotolyzed sample did not show binding of biotinyl-Bpa-mini-chaperone ( Figure 7A).

Discussion
The aAG98R mutation in aA-crystallin is associated with earlyonset cataract [19]. We and others have shown that aAG98R protein has altered structure, stability and chaperone activity [20- 22]. In vitro incubation of mutant protein at 37uC leads to the formation of soluble aggregates which, with time, coalesce and precipitate [21,22]. Almost all of the mutant protein precipitates if incubation continues for several hours at 37uC or 40uC. This behavior is typical of many mutant forms of lens crystallins, such as xD mutants L5S, V75S and I90F [32], and aB mutants F27R [33] and D140N [34]. Aggregation and precipitation are hallmarks of cataract-causing crystallin mutations. The TEM studies show that aAG98R oligomers interact with one another to form clusters of 2 to 3 oligomers or linear structures composed of 3 to 8 oligomers in 10 min of incubation at 40uC (Figure 3). At 37uC, it takes 6-8 hrs to form such aggregates, whereas at the slightly higher temperature of 40uC, aggregation begins as early as 10 min (compare Figures 3B and 3D). With time, the aggregates coalesce to form irregular aggregates having several oligomers, as shown in Figure 3E. We do not yet know which residues on the surface of the oligomers are involved in oligomer dimerization or initial aggregation. Although each subunit in the oligomer has mutation and altered structure, only a few subunits in an oligomer may have a binding interface exposed to interact with another oligomer, since all the subunits are not equally positioned due to the irregular polydisperse nature of aAG98R oligomers [21]. Such a limitation of interaction sites would initially result in a linear arrangement or the formation of dimers and trimmers of the oligomer rather than the formation of an oligomer fully decorated with additional oligomers to form a cluster of several oligomers. With time, however, the aggregates of 2-10 oligomers would interact with one another to form amorphous aggregates, as shown in images of the 30 min sample at 40uC ( Figure 3E). Because the mutant protein has an altered structure and increased hydrophobicity [20][21][22], we hypothesize that the G98R mutation exposes specific hydrophobic regions and these interact with other oligomers to form aggregates. Further studies are required to identify all of the exposed residues as a consequence of the G98R mutation. Alternately, it is possible that the increased chaperone property of the subunits in the mutant oligomer is responsible for recognizing another oligomer and this process could lead to the formation of aggregates of 2-10 oligomers. In support of this, it Proteins, 100 mg, were prepared in 400 ml of10 mM phosphate buffer, pH 7.2, and the sample temperatures were slowly raised from 25 to 48uC in 2uC steps, equilibrated for 5 min at each temperature prior to far-UV CD measurement. Molar ellipticity changes at 218 nm were plotted to determine relative stability of samples. B, Near-UV CD spectra of aAG98R and aAG98R+aA-mini-chaperone. The spectra were recorded using a protein sample of 1.5 mg/ml. The profile shown is the average of 5 scans. The far-UV CD results show that the aA-minichaperone has the stabilizing effect on aAG98R. doi:10.1371/journal.pone.0044077.g004 Figure 5. Effect of the addition of aA-crystallin-derived minichaperone on the chaperone-like activity of aAG98R against ADH aggregation. A. The aggregation of 250 mg of ADH at 43uC in 1 ml of phosphate buffer (50 mM, pH 7.2+0.15 M NaCl+10 mM EDTA) was measured in the presence and absence of aA-mini-chaperone. aAmini-chaperone (10 mg or 20 mg) was added at 70 min after initiation of heat-induced aggregation (shown by arrow) and the assay was continued for 120 min. B, Comparison of chaperone activity of aAG98R, of aAG98R stabilized with aA-mini-chaperone and of wild-type aAcrystallin. The assays were performed as described under methods using 250 mg of ADH and 50 mg of crystallins. aAG98R stabilized with aA-mini-chaperone was obtained by mixing aAG98R with aA-minichaperone and isolating the complex by TSK5000PW XL chromatography. The figures represent the typical data obtained multiple times with WT-aA-crystallin and stabilized aAG98R. The results show that the chaperone activity of aA-mini-chaperone stabilized aAG98R is comparable to that of wild-type aA-crystallin whereas the non-stabilized aAG98R has significantly reduced chaperone activity. doi:10.1371/journal.pone.0044077.g005 was shown earlier that aAG98R variant [21] and cataract-causing mutant of aA-crystallin R116C has enhanced affinity toward client proteins [35].
Earlier we discovered that a peptide representing the chaperone site of aA-crystallin is sufficient to suppress the aggregation of denaturing proteins. We showed that the peptide chaperone stabilizes the partially unfolded proteins [26][27][28][29][30] and prevents fibril formation by b-amyloid [31]. Since aAG98R has an altered structure, we investigated whether the aA-crystallin-derived minichaperone would prevent the mutant protein from precipitation during incubation. When aA-crystallin-derived mini-chaperone was incubated with aAG98R, we found that the aAG98R was stabilized and remained in solution ( Figure 2) and the stabilized aAG98R can be isolated by chromatography (Figure 1 B). This observation was confirmed by TEM study (Figure 3), which showed that aggregation of aAG98R was prevented by the aAmini-chaperone. We believe that the mini-chaperone interacts with the exposed hydrophobic regions of the mutant proteins and prevents these sites from binding to another oligomer to form aggregates that precipitate during the incubation at 37uC or 40uC. Further studies are required to confirm this since interaction of mini-chaperone with aAG98R did not result in significant change in hydrophobic probe Bis-ANS binding (Figure S3 A).
The addition of the mini-chaperone to the aAG98R sample prior to incubation at 43uC and chromatography by gel filtration increased by 8-fold the recovery of aAG98R in the soluble form ( Figure 1B). However, an inactive form of mini-chaperone (DFVPFLDVKHFSPEDLTVK) did not prevent the precipitation of aAG98R, suggesting the chaperone activity of the peptide was responsible for maintaining the mutant protein in soluble form. The interaction of the aA-crystallin-derived mini-chaperone with aAG98R was confirmed by reversed-phase HPLC analysis of the aAG98R peak recovered following incubation of active minichaperone and mutant protein ( Figure S1). Under similar experimental conditions, the wild-type aA-crystallin showed negligible interaction with mini-chaperone ( Figure S1), suggesting that the conformational change in aAG98R perhaps acted as a chaperone sensor.
Peptides substituted with the photoactive amino acid Bpa have been used to confirm the interaction between ligand and receptor [36][37][38]. We substituted one of the three phenylalanines in aAmini-chaperone with Bpa and biotinylated the N-terminal amino group to obtain biotin-DFVIFLDVKH(benzoylphenylalanine)-SPEDLTVK. The chaperone peptide was active in suppressing the aggregation of heat-denatured ADH. When the biotinyl-Bpamini-chaperone-aAG98R incubation mixture was photolyzed and subjected to SDS-PAGE and western blot analysis, covalent association of aA-mini-chaperone with aAG98R subunits was observed ( Figure 6A). The binding of biotinyl-Bpa-chaperone to aAG98R was also confirmed by MS analysis. The mass of the complex, 22592.3 m/z ( Figure 6B) is equal to 1:1 binding of aAmini-chaperone and aAG98R subunit. We found that only about 10% of the Bpa-peptide was incorporated into aAG98R subunit. Bpa photocross linking efficiency is dependent on the duration of UV exposure, the affinity of the ligands and the orientation of the Bpa residue [37]. The low insertion of Bpa in our hands may in part be due to shorter photolysis time. We did not extend the photolysis time to minimize any UV-induced structural change in the protein that may influence the interaction of peptide with aAG98R. Further, it is unlikely that all subunits in the aAG98R oligomer interact with the mini-chaperone equally because of uneven exposure of hydrophobic regions to the surface in mutant crystallin.
Modulation of wild-type a-crystallin chaperone activity by small molecules such as ATP [39], glutathione [40], arginine and aminoguanidine [41,42] has been previously reported. In those studies the modulator was used in 10-to 30-fold higher concentrations than the a-crystallin [39][40][41][42] and the conformational changes in a-crystallin in the presence of the modulator was considered to be responsible for the activity enhancement. Our study shows that 2-fold higher concentration of mini-chaperone is sufficient to stabilize the mutant aAG98R-crystallin in solution and the mini-chaperone stabilized crystallin has chaperone activity comparable to that of WT-aA-crystallin (Fig. 5B). We reported earlier that dithiothreitol (DTT) treatment of a-crystallin in the water-insoluble fraction of lens proteins restores some of the lost chaperone activity [43]. Oxidation of methionine in a-crystallin leads to loss of chaperone activity and this can be reversed by treatment with methionine sulfoxide reductase [44]. However, none of the studies carried out thus far attempted to rescue the chaperone activity in mutant forms of aAor aB-crystallins. We have previously shown that aA-crystallin-derived mini-chaperone can suppress the aggregation of several proteins [26][27][28][29][30] and prevent fibril formation by b-amyloid [31]. This study is the first report on the stabilization of a mutant aA-crystallin by a minichaperone derived fromaA-crystallin. The rescuing of chaperone activity in aAG98R by aA-mini-chaperone can be compared to the interaction of a C-terminal peptide of p53 with inactive mutant forms of the same protein and restoration of its activity [45,46]. The specific interaction between the aA-mini-chaperone and aAG98R subunit demonstrates the ability of the aA-minichaperone to act as a chaperone toward structurally perturbed aAG98R, akin to the mini-chaperone suppressing the aggregation of denaturing proteins [26,30,31].
In summary, we have shown that the aA-crystallin-derived mini-chaperone can suppress the aggregation of mutant parent protein. The increased stability of the mutant protein, coupled with only marginal increase in Rh in the presence of chaperone peptide suggests that the aA-mini-chaperone has the potential to become a therapeutic agent to stabilize the cataract-causing mutant forms of aA-crystallin. We propose that aA-crystallinderived mini-chaperones or synthetic chaperones with minichaperone electrostatic surface would have the capacity to control the aggregation of crystallin-client protein complexes or conformationally challenged proteins. Further, peptide chaperones may serve as universal chaperones for controlling diseases involving protein aggregation. Figure S1 Elution profile of mini-aA, aAG98R-miniaA complex and aA-WT treated with aA-mini-chaperone from a C8 column. 100 mg of aAG98R or WT-aA-crystallin and 10 mg of peptides were used in the study. Samples were passed through TSK5000pw column was used to separate aA-crystallin peak from the unbound peptides. The protein from the aAcrystallin peak was subsequently analyzed in a Vydac 208TP column (250 mm64.6 mm) fitted to a Shimadzu HPLC system. Acetonitrile gradient (0-80%) over a period of 40 min was used to resolve the components. Eluent A was 0.1% trifluoroacetic acid in water and eluent B was acetonitrile. Detector was set at 220 nm and the flow rate 1 ml/min. A. Analysis of aA-mini-chaperone-aAG98R and aA-mini-chaperone. B. Analysis of aA-minichaperone and WT-aA-crystallin. The HPLC analysis of the fractions at a-crystallin elution region from gel filtration column shows the binding of aA-mini-chaperone to mutant protein but not to wildtype aA-crystallin. The figure is representative of 3 independent experiments. (TIF) Figure S2 Chaperone assay in presence of either aAmini-chaperone or aA-mini-chaperone with proline substitution. The EDTA-induced aggregation of ADH assay was performed at 37uC as described under methods. In each experiment 250 mg of ADH was used. Curve 1, ADH alone; Curve 2, ADH+aA-mini-chaperone (pro) 50 mg; Curve 3, ADH+aA-min-chaperone, 50 mg. The results show that Prosubstitution abolishes the chaperone activity of mini-chaperone. The figure is representative of two independent experiments. (TIF) Figure S3 Fluorescence studies of aAG98R in presence or absence of aA-mini-chaperone. A, bis-ANS (1,19-bi(4-anilino) naphthalene-5,59-disulfonic acid) interaction with mutant protein before and after addition of aA-mini-chaperone was recorded as described under methods. The spectra shows minimal change in fluorescence after the peptide interaction with aAG98R. B, Intrinsic fluorescence spectra of aAG98R before and after addition aA-mini-chaperone. The data, representative of two independent experiments, shows minimum change in the bis-ANS binding or intrinsic tryptophan fluorescence in mutant protein following treatment with aA-mini-chaperone. (TIF)