Molecular Vision 2014; 20:1002-1016
Received 30 January 2014 | Accepted 03 July 2014 | Published 07 July 2014
1Department of Marine Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India; 2Research Reactor Institute, Kyoto University, Kumatori-cho, Sennan-gun, Osaka, Japan; 3Teikyo University, Japan; 4Department of Microbiology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India
Correspondence to: K. Anbarasu, Department of Marine Biotechnology, Bharathidasan University, Tiruchirappalli – 620 024, Tamil Nadu, India; Phone: +91 431 2407071 Etn: 436; FAX: +91 431 2407045; email: firstname.lastname@example.org
Purpose: To study the comparative structural and functional changes between wild-type (wt) and N-terminal congenital cataract causing αA-crystallin mutants (R12C, R21L, R49C, and R54C) upon exposure to different dosages of gamma rays.
Methods: Alpha A crystallin N-terminal mutants were created with the site-directed mutagenesis method. The recombinantly overexpressed and purified wt and mutant proteins were used for further studies. A 60Co source was used to generate gamma rays to irradiate wild and mutant proteins at dosages of 0.5, 1.0, and 2.0 kGy. The biophysical property of the gamma irradiated (GI) and non-gamma irradiated (NGI) αA-crystallin wt and N-terminal mutants were determined. Oligomeric size was determined by size exclusion high-performance liquid chromatography (HPLC), the secondary structure with circular dichroism (CD) spectrometry, conformation of proteins with surface hydrophobicity, and the functional characterization were determined regarding chaperone activity using the alcohol dehydrogenase (ADH) aggregation assay.
Results: αA-crystallin N-terminal mutants formed high molecular weight (HMW) cross-linked products as well as aggregates when exposed to GI compared to the NGI wt counterparts. Furthermore, all mutants exhibited changed β-sheet and random coil structure. The GI mutants demonstrated decreased surface hydrophobicity when compared to αA-crystallin wt at 0, 1.0, and 1.5 kGy; however, at 2.0 kGy a drastic increase in hydrophobicity was observed only in the mutant R54C, not the wt. In contrast, chaperone activity toward ADH was gradually elevated at the minimum level in all GI mutants, and significant elevation was observed in the R12C mutant.
Conclusions: Our findings suggest that the N-terminal mutants of αA-crystallin are structurally and functionally more sensitive to GI when compared to their NGI counterparts and wt. Protein oxidation as a result of gamma irradiation drives the protein to cross-link and aggregate culminating in cataract formation.
The human lens is composed of a unique protein known as crystallin, which is endowed with high stability and durability. Crystallin is categorized as α, β, and γ based on genetic organization and expression pattern . α-crystallin predominates in the eye lens (about 40% to 50% of the total) and is composed of two subunits, namely, αA and αB-crystallin. The chaperone-like function of α-crystallin plays a critical role in maintaining lens transparency by preventing the aggregation of unfolded proteins and cellular damage [2-4].
Cataract is defined as opacification of the eye lens associated with the breakdown of the lens microarchitecture that impairs vision . Congenital or pediatric cataract appears during birth or within the first year of life and is the most common cause of childhood blindness, which has 30% to 50% of genetic basis [6-8]. Others are intrauterine infections, uptake of drug or radiation in pregnancy, gene defects, chromosomal disorders, metabolic disease, trauma, etc. . Approximately 70% of lens opacification in congenital cataract is due to anomalies in the lens alone ; the remaining opacification includes ocular anomalies such as microphthalmia, aniridia, other anterior chamber developmental anomalies, or retinal degeneration .
Mutation in the evolutionarily conserved amino acids in the N-terminal domain of the human αA-crystallin gene provokes the formation of congenital cataract. Few such mutations were considered for this investigation. The human αA-crystallin consists of a conserved central domain ﬂanked by N- and C-terminal domains confirmed to play an important role in oligomerization [12,13]. Four congenital cataract-causing mutants in the region of the N-terminal have been predominantly reported, R12C [6,13], R21L , R49C , and R54C , worldwide. These mutant-affected members had autosomal dominant bilateral congenital nuclear cataract unveiled to possess mutation in the arginine residue. The deleterious effect on the mutational conversion of arginine reveals the crucial role of arginine in either substrate binding or protein structure stabilization thus influencing the chaperone-like activity. Most congenital mutations are mutated in the arginine residues, forming either cysteine, leucine, tryptophan, or histidine  in the eye lens αA and αB-crystallins. Modeling studies performed by Ijssell et al.  on sHSP 16.5 of Methanococcus janaschii demonstrated that the arginine residue (position 107) is buried in the hydrophobic core of the protein and forms a salt bridge with glycine. Bera et al. , in extensive structural and functional studies in the mutant R116C, reported the loss of the positive charge on the mutation in αA-crystallin that is crucial for forming salt bridges with neighboring negatively charged residues, eventually leading to protein unfolding and aggregation. Further, extensive studies have been performed to determine the structural and functional perturbation of the N-terminal mutants of αA-crystallin proteins using the recombinant approach [16,19,20], which has contributed to unraveling the mechanism of cataractogenesis.
It has been previously reported that the morphology of cataract has some correlation with the expression pattern of the mutated protein. However, varying morphologies and severities of the disease have been observed with the same mutation in different families and even within the same family suggesting the involvement of other environmental factors in the magnitude of the disease . Gamma ray–induced modification of the structural and functional properties of αA-crystallin has been performed mimicking the physiologic stress condition. However, to the best of our knowledge, there are no reports on the effect of such stress on N-terminal mutants of αA-crystallin proteins. It is crucial because proteins including the mutant forms in a cell are prone to various oxidative stresses under physiologic conditions . Constant exposure to oxidative stress, environmental elements, metabolic disorders, and genetic variations contributes to the pathomechanism of the early onset of cataract development. Apart from oxidative stress, mutations, truncations, deamidations and glycation plays a crucial role in the pathophysiology of cataractogenesis resulting in changes in the epithelial cell metabolism, lenticular protein structure, and function that are elusive . Gamma irradiation (GI)–induced oxidation in the lens closely imitates the modifications that occur in cataract formation during aging . Our previous studies demonstrate that gamma rays induce oxidation in bovine αA-crystallin evoking isomerization of Asp151, cross-link product formation, oxidation of methionine 1, and decreased chaperone activity . Thus, to understand the structural and functional behavior of the αA-crystallin N-terminal mutant proteins as a result of oxidative stress when compared to the native αA-crystallin, we adopted gamma rays as a medium for generating oxidative stress for the mutants, and the structural and functional perturbation of the mutant proteins were analyzed.
The αA-crystallin N-terminal mutants, R12C, R21L, R49C, and R54C, were created by site-directed mutagenesis using the following primers: 5′-TGG TTC AAG TGC ACC CTG GGG-3′ (forward), 5′-CCC CAG GGT GCA CTT GAA CCA-3′ (reverse) for R12C; 5′-TTC TAC CCC AGC CTG CTG TTC GAC-3′ (forward), 5′-GTC GAA CAG CAG GCT GGG GTA GAA-3′ (reverse) for R21L; 5′-CCC TAC TAC TGC CAG TCC CTC T-3′ (forward), 5′-AGA GGG ACT GGC AGT AGT AGG G-3′ (reverse) for R49C; and 5′-TCC CTC TTC TGC ACC GTG CTG-3′ (forward), 5′-CAG CAG GGT GCA GAA GAG GGA-3′ (reverse) for R54C. The primers, designed manually by replacing a single nucleotide at the middle of the primer sequence and custom synthesized at Bioserve Biotechnologies (Hyderabad, India), have cysteine and leucine in the place of the arginine codon. The cDNA construct of αA-crystallin wt in pET 3d  was used as the template for generating the mutants using the Quick Change II XL site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA) following the manufacturer’s protocol. PCR amplification was performed with an initial denaturation of 95 °C for 1 min, followed by 16 cycles of 95 °C for 50 s, 60 °C for 50 s, and 68 °C for 5 min followed by an overall extension at 68 °C for 7 min. The amplicons were digested with DpnI for 1 h at 37 °C, and the products were transformed into XL-10 Gold competent Escherichia coli cells. The transformed cells were selected on Luria bertani broth (LB) agar plates containing 50 µg/ml ampicillin. The mutant constructs were validated with restriction digestion and DNA sequence analysis.
Human αA-crystallin wt and N-terminal mutants in pET 3d vector were transformed into E. coli BL21 (DE3) pLysS cells. Protein production was induced in the cultured cells with the addition of isopropyl-1-thio-β-d-galactopyranoside (IPTG) induction to a final concentration of 0.3 mM. The cells were subsequently thawed, sonicated (six rounds of 8 pulses per minute) in 20 mM Tris-HCl, pH 7.8 containing 1 mM ethylenediamine tetraacetic acid (EDTA) and 1 mM phenyl methanesulfonyl fluoride (PMSF). The sonicated products were centrifuged at 48850 ×g for 20 min, and the soluble fraction was subject to ion exchange column chromatography on a Q Sepharose XL column (Amersham Biosciences, Piscataway, NJ) equilibrated with 20 mM Tris-HCl containing 1 mM EDTA and connected to an AKTA prime plus (GE Healthcare, Buckingham Shire, UK). The protein was eluted using a gradient of 0–1 M NaCl with a flow rate of 10 ml/min. The αA-crystallin wt and mutants were eluted at 300 mM NaCl. The purified αA-crystallin wt and mutant samples were further purified on a size exclusion column (Sephacryl 300HR; Amersham Biosciences) equilibrated with 20 mM Tris-HCl pH 7.8 containing 150 mM NaCl. The purity of the recombinant protein was checked with 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and western blotting. The purified αA-crystallin wt and its N-terminal mutants were dialyzed against 50 mM sodium phosphate buffer (pH 7.8) for further biophysical experiments.
Proteins separated using 15% SDS–PAGE were transferred onto nitrocellulose membrane at 12 V for 1 h using a semidry western blotting system (Bio-Rad, Hercules, CA). The membrane was blocked for 1 h at room temperature in Tris buffer saline with 0.1% Tween-20 (TBS-T) containing 3% (w/v) skimmed milk powder. After three washes with TBS/T of 5 min each, the membrane was incubated with polyclonal anti-αA-crystallin antibody FL-173 (Santa Cruz Biotechnology, Dallas, TX, FL-173) at a dilution of 1:1,000 overnight at 4 °C. After washing, the membrane was incubated for 1 h with anti-rabbit immunoglobulin G (IgG) conjugated to alkaline phosphatase (Sigma, St. Louis, MO) at a dilution of 1:10,000. The membrane was washed again, and the blots were developed with the bromo-4-chloro-3-idolyl phosphate/nitroblue tetrazolium chloride (BCIP/NPT) substrate (Sigma).
To investigate the effect of GI on the αA-crystallin wt and N-terminal mutants, aliquots of 1 mg/ml protein in 50 mM sodium phosphate buffer (pH 7.8) were exposed to gamma radiation doses (0.5, 1.0, and 2.0 kGy) at a rate of 509 Gy/h from a 60Co source at the Research Reactor Institute, Kyoto University (Kyoto, Japan).
The profile of the GI αA-crystallin (wt and mutant) was examined with 15% SDS–PAGE at pH 8.8 according to the standard method . The gels were stained with Coomassie brilliant blue (R250), destained and high molecular weight (HMW) cross-linked product formation of GI αA-crystallin compared to the NGI samples.
The molecular mass difference between the GI and NGI αA-crystallin wt and the congenital N-terminal mutant samples was ascertained with size exclusion chromatography on a TSKgel-G4000SWXL column (7.8 × 300 mm, Tosho, Tokyo, Japan). The column was equilibrated in 50 mM Tris-HCl containing 150 mM NaCl with a flow rate of 0.8 ml/min. About 100 µl of the 1 mg/ml experimental protein was injected onto the column, and the elution protein profile was recorded with absorbance detection at 280 nm. Molecular masses were acquired by calibrating the column with molecular mass standards.
A circular dichroism (CD) spectropolarimeter (Jasco 810, Kyoto, Japan) was used to record the CD spectra of the GI and NGI αA-crystallin wt and N-terminal mutants. Far-ultraviolet (UV) CD measurements were performed between wavelengths of 195 to 250 nm at room temperature. Scans were performed using a cylindrical quartz cuvette with a 1 mm path length. Protein samples of 0.1 mg/ml were prepared in 50 mM sodium phosphate buffer (pH 7.8). Spectra represented are the average of five scans. The buffer signal was subtracted and smoothed.
The relative surface hydrophobicity of the GI and NGI αA-crystallin wt and N-terminal mutants were determined with 4,4'-bis(1-anilinonaphthalene 8-sulfonate) (bis-ANS), a hydrophobic probe, as described previously . Briefly, an aliquot of 100 µl of bis-ANS (0.1 mg/ml) was added to 100 µl of protein (1 mg/ml) along with 800 µl of 50 mM sodium phosphate buffer (pH 7.8). Before the experiment, the reaction mixtures were incubated at room temperature for 15 min in the dark. The intensity of the fluorescence was measured using an F-4500 Hitachi fluorescence spectrophotometer (Hitachi, Tokyo, Japan) with excitation of 390 nm and emission at 420–560 nm.
Chaperone activities of the GI and NGI αA-crystallin wt and N-terminal mutants were measured via the ability of crystallin to protect against the aggregation of alcohol dehydrogenase (ADH) induced by EDTA with αA-crystallin and ADH in a 1:5 ratio (w/w). An aliquot (500 μl) of 0.5 mg/ml ADH and 350 μl of 50 mM sodium phosphate buffer (pH 7.2) containing 100 mM NaCl were incubated for 30 min at 37 °C with 50 μl of 1.0 mg/ml GI or NGI αA-crystallin wt and N-terminal mutants. ADH aggregation was initiated by adding 100 μl of 100 mM EDTA to each of the incubated reaction mixtures in a thermostat holder, and light scattering monitored for 60 min at 360 nm with a ultraviolet-visible (UV-Vis) spectrophotometer with a thermostat controller (Shimadzu UV-1200, Kyoto, Japan). The data were statistically analyzed with one-way ANOVA using SPSS software version 16 (Chicago, IL), and significance was considered at the level of *p<0.05 and *** p<0.01.
The point mutants, created by site-directed mutagenesis and preliminarily confirmed with DNA double-digestion, are shown in Figure 1A demonstrating clear and distinct bands at approximately 512 bp and 4.6 kb on a 2% agarose gel, confirming the presence of the insert and the vector, respectively. Further, Figure 2 shows the confirmation and comparison of the undigested plasmid and double-digested clones harboring the mutants R12C, R21L, R49C, and R54C. Figure 1B shows the chromatogram of the clones obtained as a result of sequencing. The mutated sequence in the chromatogram of the clones, R12C, R21L, R49C, and R54C, was analyzed and confirmed.
The sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) results demonstrate a level of purity of the eluted proteins of more than 95% (Figure 1C), which was further confirmed with western blotting (Figure 1D). The blotting results show the appearance of insoluble dark blue diformazan precipitate thus confirming the presence of αA-crystallin and mutants.
Figure 3 indicates a decrease in monomeric forms and an increase in cross-linked product formation in all N-terminal mutants as the gamma rays dosage increased, when compared to the wild-type and NGI counterparts. αA-crystallin wt showed a decrease in the monomeric form and slightly increased higher cross-linked product formation at 2.0 kGy. However, the mutants R21L and R54C exhibited a drastic decrease in the monomeric form and increased high molecular weight (HMW) cross-linked product formation at all dosages of GI. The mutants R12C and R49C demonstrated a decrease in the monomeric form, and increased HMW cross-linked products started to be detectable upon exposure to 1.0 kGy and above with a significant level at 2.0 kGy of GI. The αA-crystallin wt and the N-terminal mutants formed HMW cross-linked products due to GI, but the level of product formation was higher in the mutants compared to the wt and NGI mutants. In a nutshell, all mutants started to form cross-linked product at a minimal dosage of 0.5 kGy; nevertheless, in αA-crystallin wt, the first sign of a significant level of increased cross-linked product formation was observed only at 2.0 kGy of GI. The densitometric analysis also confirmed the diminishing level of monomeric form due to GI in the N-terminal mutants analyzed with ImageJ software (Figure 4).
Figure 5A–C shows no significant changes in the elution profile of the NGI and GI αA-crystallin wt up to 1.0 kGy. Moreover, at the lower irradiation dosage of 0.5 kGy the elution profile of the NGI and GI αA-crystallin wt and N-terminal mutants were comparable. However, the elution peaks of the N-terminal mutants R12C, R49C, and R54C at 1.0 kGy appeared earlier than the control, demonstrating an increased size due to protein aggregation. Furthermore, at 2.0 kGy the mutant proteins elution profile was near the void volume corresponding to an apparent molecular mass of >7 × 106 Da (Figure 5D). The R21L mutant showed an increased aggregate size only at 2.0 kGy of gamma irradiation. Thus, it can be concluded that the recombinant αA-crystallin wt appears stable to GI whereas the N-terminal mutants started forming aggregation products from 1.0 kGy. Thus, mutation in arginine to cysteine conversion enhances cross-linked product formation when compared to the leucine conversion as well as the formation of disulfide bond between intra- or inter-polypeptide.
Far-UV spectra indicated that gamma irradiation of mutant proteins perturbed the secondary structure (Figure 6). The NGI αA-crystallin wt and N-terminal mutants almost retained their secondary structure without many changes, and a slight change was observed only in the R54C. Similarly, the αA-crystallin wt retained its secondary structure even at 2.0 kGy of GI (Figure 6A–D). In contrast, GI altered the β-sheet content and increased the random coil structure after 0.5 kGy in all N-terminal mutants. The greatest change in the β-sheet and random coil structure was observed in the mutants R12C, R21L, and R49C after 0.5 kGy exposure whereas the R54C mutant exhibited only slight changes in the β-sheet structure. We found that the degree of change or increase was directly proportional to the irradiation dosage and the location of the point mutation; the higher GI dosage resulted in greater perturbation in the secondary structure of the αA-crystallin N-terminal mutants. At 2.0 kGy, a drastic perturbation of the secondary structure was observed for all mutant proteins (Figure 6D).
Figure 7 shows the bis-ANS fluorescence intensity of the NGI and GI αA-crystallin wt and N-terminal mutants. At 0, 0.5, and 1.0 kGy, all the mutants exhibited decreased surface hydrophobicity when compared to the αA-crystallin wt (Figure 7A–C). At 1.0 kGy, when compared to the NGI counterparts, the αA-crystallin wt and the mutant R12C exhibited a considerable decrease in surface hydrophobicity. Unexpectedly, R54C demonstrated a sudden increase in surface hydrophobicity at 2.0 kGy, and αA-crystallin wt, R21C, and R49C did not show any changes in fluorescence. However, a slight decrease in surface hydrophobicity was observed in the mutant R21L (Figure 7D).
The chaperone function of the GI and NGI αA-crystallin wt and mutant proteins was determined using the client protein alcohol dehyderogenase (ADH). The chaperone activity of the αA-crystallin wt was slightly decreased after GI (Figure 8). The chaperone activity for all mutants irradiated with gamma rays against ADH as the substrate is given in Table 1 in terms of percentage. Notably, all GI N-terminal mutants exhibited an increase in chaperone activity compared to the NGI mutant counterparts as observed by the increasing suppression of ADH aggregation. As shown in Table 1, at 0 kGy the αA-crystallin wt showed 92% of chaperone activity; however, the mutants R12C, R21L, R49C, and R54C exhibited only 46.7%, 72.3%, 87.8%, and 78% of chaperone activity, respectively. Nevertheless, at 0.5 kGy GI all mutants, R12C, R21L, R49C, and R54C exhibited a slight increase in chaperone activity of 61.4%, 84.8%, 93.5%, and 83.6%, respectively, whereas the αA-crystallin wt showed s non-significant meager loss in its chaperone activity, exhibiting 91.3%. At 1.0 and 2.0 kGy, the αA-crystallin wt demonstrated 89.2% and 87.9% of chaperone activity, respectively; however, all mutants showed increased chaperone activity. R12C, R21L, R49C, and R54C exhibited 73.2%, 89.8%, 95.9%, and 85.7% of chaperone activity, respectively, at 1.0 kGy, and at 2.0 kGy, the rates were 80.1%, 92.1%, 97.4%, and 88.7%, respectively. The results clearly indicate the significance of the arginine residue in the N-terminal domain of αA-crystallin, and GI changed the chaperone function in the wt and mutants. Moreover, the arginine residues play a vital role in chaperone activity. The increased chaperone activity of the GI N-terminal mutants may be due to changes in hydrophobicity, the increased number and position of cysteine residues, the involvement of disulfide linkage formation, covalent protein–protein interaction, substrate binding potential, etc.
αA-crystallin wt is reported to be more resistant to the effects of gamma ray–induced oxidative damage than αB-wt . Of the various post-translational modifications reported previously [26-32], oxidation of αA-crystallin leads to altered biophysical properties such as oligomerization, secondary structure, chaperone activity, and hydrophobicity of the protein. Gamma rays have the ability to induce reactive oxygen species (ROS) production in aqueous solution and thus can result in oxidization of the first αA-crystallin methionine residue to methonine sulfoxide, tryptophan to hydroxytryptophan, and N-formylkynurenine  and aspartic acid residues at positions 68 and 151 to D-β, D-α, and L-β-aspartic acid . However, little is known about the effect of oxidation on the structural and functional parameters of N-terminal mutants; thus, this study is the first of its kind.
An intriguing finding of the present study is that the αA-crystallin N-terminal mutants (R12C, R21L, R49C, and R54C) exhibited altered structural and functional properties even at 0.5 kGy of GI, and significant levels of modification were recorded at 1.0 kGy and 2.0 kGy exposure. The effect of GI on the oligomeric size of the αA-crystallin wt and N-terminal mutants examined with size exclusion high-performance liquid chromatography (HPLC) demonstrated an increase in the heterogeneity of aggregation in the R12C, R49C, and R54C mutants compared to the NGI mutant counterparts. Significantly, the mutant R21L was resistant to GI at low dosages and showed meager notable aggregation only at 2.0 kGy of GI exposure. It was ascertained that the αA-crystallin N-terminal mutants were more sensitive to GI as a result of changes in the native structure leading to the formation of HMW aggregates as described earlier [35-37]. There was an increase in the aggregation size of the GI N-terminal mutants, since the N-terminal domain maintains the oligomeric size of αA-crystallin , a mutation in this domain may make the protein more prone to oxidation. The formation of HMW aggregates as a result of GI may be due to the formation of inter-or intrapeptide cross-linking or hydrophobic as well as disulfide bond (S-S) formation by the amino acid radical formed within a peptide chain with the amino radical in another peptide [16,39-42]. Moreover, the size exclusion HPLC findings demonstrate that mutant proteins that have more cysteine residue enhance the formation of HMW aggregate eventually contributing to lens opacification thus leading to scattering of the light. However, αA-crystallin wt and R21L were relatively stable up to 1.0 kGy although a slight increase in aggregate size was observed on exposure at 2.0 kGy.
ROS-induced oxidation results in the disruption of amino acid residue side chains, cleavage of peptide bonds, and formation of covalent protein–protein cross-linked derivatives . In this study, the ROS generated by GI at a low dosage of 0.5 kGy displayed a slight breakdown in the polypeptide chain as revealed in the SDS–PAGE analysis. It increased significantly when exposed to higher dosages. At 1.0 kGy exposure, all the N-terminal mutant proteins were aggregated such that they were unable to penetrate the separating gel. The high amount of cross-linked product formation in the N-terminal mutants revealed their increased susceptibility to ROS-induced oxidation compared to the αA-crystallin wt. Moreover, the sulfur-containing and aromatic amino acids react highly with free radicals compared to aliphatic amino acids . The aromatic amino acid tyrosine is highly sensitive to free radicals leading to hydrogen abstraction resulting in the formation of tyrosyl (phenoxyl) radicals and readily reacts with other tyrosyl molecules to form bityrosine . The intermolecular bityrosine is the mechanism behind the protein aggregation in addition to other cross-links of the proteins . Our results are in line with previous reports that radiolysis of protein extends to form protein–protein cross-linking through tyrosine-tyrosine and nondisulfide cross-links . Eventually, at a high dosage of 2.0 kGy, the increased molecular weight of the mutant proteins was found indicating the formation of cross-linked products of the degraded protein molecules .
Furthermore, GI of the αA-crystallin N-terminal mutants resulted in significant changes in the secondary structural properties with changes in the β-sheet as well as random coil structural conformation, relative to the NGI counterparts. In contrast, the αA-crystallin wt almost retained its secondary structural integrity up to 2.0 kGy of GI, which is consistent with our previous report, in which GI up to 4.0 kGy did not affect the secondary structure of the αA-crystallin wt . As shown in Figure 6, irradiation affected the CD spectrum of the mutant protein disrupting its fold. Irradiation primarily decreased the ordered structure of the mutant proteins, which resulted in an altered β-sheet structure with a concomitant increase in the random coil content. This shows that the secondary structure of the αA-crystallin N-terminal mutants is more sensitive to gamma rays. The change in the secondary structure is attributed to the breakdown of the covalent bonds and the disruption of the ordered structure of proteins [42,47] finally altering the native function of the protein. The bis-ANS assay results demonstrate that the GI N-terminal mutants R12C and R54C exhibited unusual behavior in exposing hydrophobic patches to the environment. At 0.5 kGy, the mutants R21L and R54C exhibited decreased surface hydrophobicity retaining the same on exposure to 1.0 kGy; however, at 2.0 kGy, R54C demonstrated significant increased fluorescence, and R21L demonstrated decreased fluorescence. R49C in contrast to the other mutants exhibited a slight decrease in surface hydrophobicity at 1.0 and 2.0 kGy. Our results are in contrast to earlier reports that demonstrated in vitro treatment of purified proteins with radiolytically generated hydroxyl radicals leads to an increase in hydrophobicity [39,40,48]. In this study, we have reported a substantial increase in hydrophobicity at low dosage and a decrease at higher dosage upon gamma irradiation of αA-crystallin N-terminal mutants. A plausible explanation for the discrepancy could be that the presence of the mutations in different regions of the N-terminal domain results in different behavior and characteristics, and thus, we would not expect all mutants to undergo all expected changes . Taking into account the size exclusion, SDS–PAGE, surface hydrophobicity as well as CD experimental results, we concluded that gamma irradiation markedly affects the structural characteristics of all αA-crystallin N-terminal mutants.
Even though there were differences in the exposure of the hydrophobic surface in all mutants after GI, the chaperone activity was increased slightly upon GI revealing that there is no stringent relationship between surface hydrophobicity and the chaperone function in α-crystallin as reported earlier [49,50]. The molecular chaperone function of α-crystallin is known to prevent protein unfolding and aggregation. Many factors including micronutrients are known to have the ability to alter the chaperone function of αA-crystallin , although the molecular mechanisms behind the chaperone activity are not completely established. In general, the chaperone activity of αA-crystallin may decrease due to oxidative stress, deamidation, truncation, etc. Since chaperone activity is one of the major functions of αA-crystallin, a reliable and standardized method was used to quantify the chaperone activity of the GI N-terminal mutants in this study. Specifically, EDTA-induced denaturation of ADH was exploited. The major grounds for excluding other substrates such as βL, γD crystallins, and insulin are the highly reproducible outcomes obtained with ADH . An essential criterion in chaperone function is the extent of binding of the chaperone with the substrate. We found that the NGI αA-crystallin wt, mutants R49C and R54C exhibited nearly complete suppression of ADH aggregation whereas the ability was lost in the mutants R12C and R21L corroborating with Kore et al.’s results . However, there was marked elevation in chaperone activity upon oxidation of crystallin protein in all N-terminal mutants relative to NGI counterparts. In particular, the R12C mutant (NGI) lost 50% of chaperone activity due to a mutation as described earlier  but showed a fold increase in activity upon gamma irradiation. We speculated that the increased chaperone activity might be due to changes in hydrophobicity, as a result of oxidation induced by GI, because previously Jang et al.  reported a positive correlation between hydrophobicity and chaperone activity. However, we found that in the case of the GI N-terminal mutants of αA-crystallin, hydrophobicity was not always related to chaperone activity as concluded by several other researchers [50,53]. Consequently, gamma ray exposure modifies other physical properties of the mutant proteins rather than hydrophobicity, which increased the chaperone function (yet to be revealed) of the mutated protein . Further, the increase in chaperone activity may be attributed to increased number of cysteine residues at the 12th position involved in disulfide bond formation and covalent protein–protein interaction, which resulted in increased substrate binding potential of the GI mutant proteins. A vibronic transition of chaperone activity in R12C was observed on GI, which shows that this mutant is more sensitive to oxidation resulting in increased substrate binding potential. However, it is far-fetched to claim that irradiation increases the stability of these mutations because the SDS–PAGE profile, size exclusion chromatogram, and far-UV CD results depict the formation of cross-linked products, large aggregates, and secondary structural alterations. Moreover, it remains questionable whether the gained chaperone function of the protein could prevent the formation of aggregation of other mutant/target proteins or not.
In conclusion, this report analyzed the comparative structural perturbation and functional changes of αA-crystallin wt and its N-terminal mutants in response to gamma irradiation at different dosages. The results demonstrate that an increasing dosage of gamma irradiation resulted in highly altered structural and functional properties of the proteins. High molecular weight cross-linked product formation and aggregation were observed in the mutants exposed to GI reflecting the occurrence of protein oxidation in addition to changes in the surface hydrophobicity pattern. These data clearly indicate that all N-terminal mutants are structurally sensitive to GI. Further, compared with the results of the structural analyses, we speculate that the GI αA-crystallin mutant proteins might have been truncated or oxidized and formed a new complex structure with a changed physical nature that culminated in increased chaperone activity. However, extended studies are needed to elucidate the mechanism by which oxidation induced by GI results in N-terminal mutants’ gain in chaperone activity.
The authors gratefully acknowledge the financial assistance from Department of Science and Technology (DST), New Delhi as DST-YS-SR/FT/LS-153/2008/(2010-2013) and DST-JSPS-(DST/INT/JSPS/P-119/ (2011-2013) sanctioned to the corresponding author (Dr. K.A.). The 4th authors thank DST-INSPIRE for senior research fellowship. Authors thank all the anonymous reviewer’s for their valuable comments to improve the quality of this manuscript.