Differential protective activity of alpha A- and alphaB-crystallin in lens epithelial cells

alphaA- and alphaB-crystallins are molecular chaperones expressed at low levels in lens epithelial cells, and their expression increases dramatically during differentiation to lens fibers. However, the functions of alphaA- and alphaB-crystallins in lens epithelial cells have not been studied in detail. In this study, the relative ability of alphaA- and alphaB-crystallin, in protecting lens epithelial cells from apoptotic cell death was determined. The introduction of alphaA-crystallin in the transformed human lens epithelial (HLE) B-3 lens epithelial cell line (which expresses low endogenous levels of alphaB-crystallin) led to a nearly complete protection of cell death induced by staurosporine, Fas monoclonal antibody, or the cytokine tumor necrosis factor alpha. To further study the relative protective activities of alphaA- and alphaB-crystallins, we created a cell line derived from alphaA-/-alphaB-/- double knockout mouse lens epithelia by infecting primary cells with Ad12-SV40 hybrid virus. The transformed cell line alphaAalphaBKO1 derived from alphaA/alphaB double knockout cells was transfected with alphaA- or alphaB-crystallin cDNA contained in pCIneo mammalian expression vector. Cells expressing different amounts of either alphaA-crystallin or alphaB-crystallin were isolated. The ability of alphaA- or alphaB-crystallin to confer protection from apoptotic cell death was determined by annexin labeling and flow cytometry of staurosporine- or UVA- treated cells. The results indicate that the anti-apoptotic activity of alphaA-crystallin was two to three-fold higher than that of alphaB-crystallin. Our work suggests that comparing the in vitro annexin labeling of lens epithelial cells is an effective way to measure the protective activity of alphaA- and alphaB-crystallin. Since the expression of alphaA-crystallin is largely restricted to the lens, its greater protective effect against apoptosis suggests that it may play a significant role in protecting lens epithelial cells from stress.     

Temperature-dependent chaperone activity and structural properties of human alphaA- and alphaB-crystallins

The chaperone activity and biophysical properties of recombinant human alphaA- and alphaB-crystallins were studied by light scattering and spectroscopic methods. While the chaperone function of alphaA-crystallin markedly improves with an increase in temperature, the activity of alphaB homopolymer appears to change very little upon heating. Compared with alphaB-crystallin, the alphaA-homopolymer is markedly less active at low temperatures, but becomes a more active species at high temperatures. At physiologically relevant temperatures, the alphaB homopolymer appears to be modestly (two times or less) more potent chaperone than alphaA homopolymer. In contrast to very similar thermotropic changes in the secondary structure of both homopolymers, alphaA- and alphaB-crystallins markedly differ with respect to the temperature-dependent surface hydrophobicity profiles. Upon heating, alphaA-crystallin undergoes a conformational transition resulting in the exposure of additional hydrophobic sites, whereas no such transition occurs for alphaB-crystallin. The correlation between temperature-dependent changes in the chaperone activity and hydrophobicity properties of the individual homopolymers supports the view that the chaperone activity of alpha-crystallin is dependent on the presence of surface-exposed hydrophobic patches. However, the present data also show that the surface hydrophobicity is not the sole determinant of the chaperone function of alpha-crystallin.     

Insights into hydrophobicity and the chaperone-like function of alphaA- and alphaB-crystallins: an isothermal titration calorimetric study

Alpha-crystallin, composed of two subunits, alphaA and alphaB, has been shown to function as a molecular chaperone that prevents aggregation of other proteins under stress conditions. The exposed hydrophobic surfaces of alpha-crystallins have been implicated in this process, but their exact role has not been elucidated. In this study, we quantify the hydrophobic surfaces of alphaA- and alphaB-crystallins by isothermal titration calorimetry using 8-anilino-1-napthalenesulfonic acid (ANS) as a hydrophobic probe and analyze its correlation to the chaperone potential of alphaA- and alphaB-crystallins under various conditions. Two ANS binding sites, one with low and another with high affinity, were clearly detected, with alphaB showing a higher number of sites than alphaA at 30 degrees C. In agreement with the higher number of hydrophobic sites, alphaB-crystallin demonstrated higher chaperone activity than alphaA at this temperature. Thermodynamic analysis of ANS binding to alphaA- and alphaB-crystallins indicates that high affinity binding is driven by both enthalpy and entropy changes, with entropy dominating the low affinity binding. Interestingly, although the number of ANS binding sites was similar for alphaA and alphaB at 15 degrees C, alphaA was more potent than alphaB in preventing aggregation of the insulin B-chain. Although there was no change in the number of high affinity binding sites of alphaA and alphaB for ANS upon preheating, there was an increase in the number of low affinity sites of alphaA and alphaB. Preheated alphaA, in contrast to alphaB, exhibited remarkably enhanced chaperone activity. Our results indicate that although hydrophobicity appears to be a factor in determining the chaperone-like activity of alpha-crystallins, it does not quantitatively correlate with the chaperone function of alpha-crystallins.     

Domain swapping in human alpha A and alpha B crystallins affects oligomerization and enhances chaperone-like activity

alphaA and alphaB crystallins, members of the small heat shock protein family, prevent aggregation of proteins by their chaperone-like activity. These two proteins, although very homologous, particularly in the C-terminal region, which contains the highly conserved "alpha-crystallin domain," show differences in their protective ability toward aggregation-prone target proteins. In order to investigate the differences between alphaA and alphaB crystallins, we engineered two chimeric proteins, alphaANBC and alphaBNAC, by swapping the N-terminal domains of alphaA and alphaB crystallins. The chimeras were cloned and expressed in Escherichia coli. The purified recombinant wild-type and chimeric proteins were characterized by fluorescence and circular dichroism spectroscopy and gel permeation chromatography to study the changes in secondary, tertiary, and quaternary structure. Circular dichroism studies show structural changes in the chimeric proteins. alphaBNAC binds more 8-anilinonaphthalene-1-sulfonic acid than the alphaANBC and the wild-type proteins, indicating increased accessible hydrophobic regions. The oligomeric state of alphaANBC is comparable to wild-type alphaB homoaggregate. However, there is a large increase in the oligomer size of the alphaBNAC chimera. Interestingly, swapping domains results in complete loss of chaperone-like activity of alphaANBC, whereas alphaBNAC shows severalfold increase in its protective ability. Our findings show the importance of the N- and C-terminal domains of alphaA and alphaB crystallins in subunit oligomerization and chaperone-like activity. Domain swapping results in an engineered protein with significantly enhanced chaperone-like activity.     

Distinct roles of alphaA- and alphaB-crystallins under thermal and UV stresses

alpha-Crystallin, a major protein of all vertebrate lenses, consists of two subunits, alphaA and alphaB, which form polymeric aggregates with an average molecular mass of about 800kDa. In this study, we have employed various biophysical methods to study aggregate sizes and conformational properties of purified alphaA, alphaB subunits, and cloned recombinant alphaB subunit. From far- and near-UV CD spectra, native alpha-, alphaA-, alphaB-, and recombinant alphaB-crystallins from porcine lenses all show similar beta-sheet conformation to that from bovine and human lenses as reported previously. By means of gel-filtration chromatography and dynamic light scattering, we have found that the molecular sizes of all four crystallin aggregates are polydispersedly distributed in the following order of aggregate sizes, i.e., native alpha>alphaA>alphaB approximately recombinant alphaB. To investigate the structural and functional relationships, we have also compared the chaperone activities of all four alpha-crystallin aggregates at different temperatures. From the results of chaperone-activity assays, ANS (8-anilinonaphthalene-1-sulfonic acid) binding and thermal stability studies, there appeared to be at least two factors playing major roles in the chaperone-like activity of these lens proteins: one is the hydrophobicity of the exposed protein surface and the other is the structural stability associated with each protein. We showed that alphaA-crystallin is a better chaperone to protect gamma-crystallin against UV irradiation than alphaB-crystallin, in contrast to the observation that alphaB is generally a better chaperoning protein than alphaA for enzyme protective assays at physiological temperatures.     

Thermodynamic stability of human lens recombinant alphaA- and alphaB-crystallins

Lens alpha-crystallin is a 600-800-kDa heterogeneous oligomer protein consisting of two subunits, alphaA and alphaB. The homogeneous oligomers (alphaA- and alphaB-crystallins) have been prepared by recombinant DNA technology and shown to differ in the following biophysical/biochemical properties: hydrophobicity, chaperone-like activity, subunit exchange rate, and thermal stability. In this study, we studied their thermodynamic stability by unfolding in guanidine hydrochloride. The unfolding was probed by three spectroscopic parameters: absorbance at 235 nm, Trp fluorescence intensity at 320 nm, and far-UV circular dichroism at 223 nm. Global analysis indicated that a three-state model better describes the unfolding behavior than a two-state model, an indication that there are stable intermediates for both alphaA- and alphaB-crystallins. In terms of standard free energy (DeltaG(NU)(H(2)(O))), alphaA-crystallin is slightly more stable than alphaB-crystallin. The significance of the intermediates may be related to the functioning of alpha-crystallins as chaperone-like molecules.     

Elevated expression of alphaA- and alphaB-crystallins in streptozotocin-induced diabetic rat

alpha-Crystallin, a predominant protein of the ocular lens, is composed of two subunits, alphaA and alphaB. Of these, alphaB-crystallin has been shown to present widely in non-lenticular tissues while alphaA-crystallin is largely lens-specific. Although, expression of alphaB-crystallin is elevated under various stress and pathological conditions, yet its physiological significance remained unknown. Some studies suggest that the expression of alphaB-crystallin gene is related to oxidative stress. Persistent hyperglycemia during uncontrolled diabetes is known to cause oxidative stress, which has been implicated in various secondary complications of diabetes. Hence, expression of alphaA- and alphaB-crystallins in various tissues of streptozotocin (STZ)-induced diabetic Wistar-NIN rats was investigated by RT-PCR and immunoblotting. While expression of alphaB-crystallin was noted in the wide range of tissues examined in the study, alphaA-crystallin expression was detected only in lens and retina. Interestingly, alphaB-crystallin expression was elevated in lens, heart, muscle, and brain, but decreased in adipose tissue of diabetic rats compared to control rats. alphaA-Crystallin expression was increased in retina of diabetic rat. Increased oxidative stress appears to be a major stimulus for the enhanced expression of alphaA- and alphaB-crystallins in the tissues of diabetic rats and elevated expression of alpha-crystallin may have a protective role against metabolic stress. Interestingly, feeding of curcumin, a dietary antioxidant, to diabetic rats attenuated the enhanced expression of alphaB-crystallin. The results indicate that elevated expression of alpha-crystallins in some tissues may have implications in pathophysiology of diabetic complications.     

alphaA- and alphaB-crystallins protect glucose-6-phosphate dehydrogenase against UVB irradiation-induced inactivation

alpha-Crystallin, a major eye lens protein, has been shown to function like a molecular chaperone by suppressing the aggregation of other proteins induced by various stress conditions. Ultraviolet (UV) radiation is known to cause structural and functional alterations in the lens macromolecules. Earlier we observed that exposure of rat lens to in vitro UV radiation led to inactivation of many lens enzymes including glucose-6-phosphate dehydrogenase (G6PD). In the present paper, we show that alpha-crystallin (alphaA and alphaB) protects G6PD from UVB irradiation induced inactivation. While, at 25 degrees C, there was a time-dependent decrease in G6PD activity upon irradiation at 300 nm, at 40 degrees C there was a complete loss of activity within 30 min even without irradiation. The loss of activity of G6PD was prevented significantly, if alphaA- or alphaB-crystallin was present during irradiation. At 25 degrees C, alphaB-crystallin was slightly a better chaperone in protecting G6PD against UVB inactivation. Interestingly, at 40 degrees C, alphaA- and alphaB-crystallins not only prevent the loss of G6PD activity but also protect against UVB inactivation. However, alphaA- and alphaB-crystallins were equally efficient at 40 degrees C in protecting G6PD.     

In vitro dephosphorylation of alpha-crystallin is dependent on the state of oligomerization

alphaA- and alphaB-crystallin, members of the small heat shock protein family, are present in lens cell extracts as large aggregates. Both alpha-crystallins are found partially phosphorylated. This study tests the ability of five phosphatases (protein phosphatase PP1, PP2A, PP2B, alkaline and acid phosphatases) to dephosphorylate alphaA- and alphaB-crystallin in vitro. Activity of a phosphatase was dependent on the size of the aggregate. Each of the phosphatases tested showed different specificity and efficiency towards alphaA- and alphaB-crystallins, which depended on the oligomeric state of the alpha-crystallin aggregate. Alkaline phosphatase dephosphorylated both alphaA- and alphaB-crystallin. The reaction was faster when alpha-crystallin was in a tetrameric form. PP2A dephosphorylated primarily alphaA-crystallin but only after the conversion of alpha-crystallin to tetramers. PP1 and PP2B did not dephosphorylate either alphaA- or alphaB-crystallins present as large aggregates but could not be tested on the lower molecular weight form of alphaA-crystallin. Acid phosphatase dephosphorylated both alphaA- and alphaB-crystallin. The results suggest that an important relationship exists between the structure of alpha-crystallin and its level of phosphorylation in the cell.     

Differential temperature-dependent chaperone-like activity of alphaA- and alphaB-crystallin homoaggregates

alpha-Crystallin, a heteromultimeric protein made up of alphaA- and alphaB-crystallins, functions as a molecular chaperone in preventing the aggregation of proteins. We have shown earlier that structural perturbation of alpha-crystallin can enhance its chaperone-like activity severalfold. The two subunits of alpha-crystallin have extensive sequence homology and individually display chaperone-like activity. We have investigated the chaperone-like activity of alphaA- and alphaB-crystallin homoaggregates against thermal and nonthermal modes of aggregation. We find that, against a nonthermal mode of aggregation, alphaB-crystallin shows significant protective ability even at subphysiological temperatures, at which alphaA-crystallin or heteromultimeric alpha-crystallin exhibit very little chaperone-like activity. Interestingly, differences in the protective ability of these homoaggregates against the thermal aggregation of beta(L)-crystallin is negligible. To investigate this differential behavior, we have monitored the temperature-dependent structural changes in both the proteins using fluorescence and circular dichroism spectroscopy. Intrinsic tryptophan fluorescence quench-ing by acrylamide shows that the tryptophans in alphaB-crystallin are more accessible than the lone tryptophan in alphaA-crystallin even at 25 degrees C. Protein-bound 8-anilinonaphthalene-1-sulfonate fluorescence demonstrates the higher solvent accessibility of hydrophobic surfaces on alphaB-crystallin. Circular dichroism studies show some tertiary structural changes in alphaA-crystallin above 50 degrees C. alphaB-crystallin, on the other hand, shows significant alteration of tertiary structure by 45 degrees C. Our study demonstrates that despite a high degree of sequence homology and their generally accepted structural similarity, alphaB-crystallin is much more sensitive to temperature-dependent structural perturbation than alphaA- or alpha-crystallin and shows differences in its chaperone-like properties. These differences appear to be relevant to temperature-dependent enhancement of chaperone-like activity of alpha-crystallin and indicate different roles for the two proteins both in alpha-crystallin heteroaggregate and as separate proteins under stress conditions.     

AlphaA-crystallin interacting regions in the small heat shock protein, alphaB-crystallin

Amino acid sequences of alphaB-crystallin, involved in interaction with alphaA-crystallin, were determined by using peptide scans. Positionally addressable 20-mer overlapping peptides, representing the entire sequence of alphaB-crystallin, were synthesized on a PVDF membrane. The membrane was blocked with albumin and incubated with purified alphaA-crystallin. Probing the membrane with alphaA-crystallin-specific antibodies revealed residues 42-57, 60-71, and 88-123 in alphaB-crystallin to interact with alphaA-crystallin. Residues 42-57 and 60-71 interacted more strongly with alphaA-crystallin than the 88-123 sequence of alphaB-crystallin. Binding of one of the alphaB peptides (42-57) to alphaA-crystallin was also confirmed by gel filtration studies and HPLC analysis. The alphaB-crystallin sequences involved in interaction with alphaA-crystallin were distinct from the chaperone sites reported earlier as binding of the alphaB sequence from residues 42-57 does not alter the chaperone-like function of alphaA-crystallin. To identify the critical residues involved in interaction with alphaA-crystallin, R50G and P51A mutants of alphaB-crystallin were made and tested for their ability to interact with alphaA-crystallin. The oligomeric size and hydrophobicity of the mutants were similar. Circular dichroism studies showed that the P51A mutation increased the alpha-helical content of the protein. While the alphaBR50G mutant showed chaperone-like activity similar to wild-type alphaB, alphaBP51A showed reduced chaperone function. Fluorescence resonance energy transfer studies showed that the P51A mutation decreased the rate of subunit exchange with alphaA by 63%, whereas the R50G mutation reduced the exchange rate by 23%. Similar to wild-type alphaB, alphaB-crystallin peptide (42-57) effectively competed with alphaBP51A and alphaBR50G for interaction with alphaA. Thus, our studies showed that the alphaB-crystallin sequence (42-57) is one of the interacting regions in alphaB and alphaA oligomer formation.     

Deamidation affects structural and functional properties of human alphaA-crystallin and its oligomerization with alphaB-crystallin

To determine the effects of deamidation on structural and functional properties of alphaA-crystallin, three mutants (N101D, N123D, and N101D/N123D) were generated. Deamidated alphaB-crystallin mutants (N78D, N146D, and N78D/N146D), characterized in a previous study (Gupta, R., and Srivastava, O. P. (2004) Invest. Ophthalmol. Vis. Sci. 45, 206-214) were also used. The biophysical and chaperone properties were determined in (a) homoaggregates of alphaA mutants (N101D, N123D, and N101D/N123D) and (b) reconstituted heteroaggregates of alpha-crystallin containing (i) wild type alphaA (WT-alphaA): WT-alphaB crystallins, (ii) individual alphaA-deamidated mutants:WT-alphaB crystallins, and (iii) WT-alphaA:individual alphaB-deamidated mutant crystallins. Compared with the WT-alphaA, the three alphaA-deamidated mutants showed reduced levels of chaperone activity, alterations in secondary and tertiary structures, and larger aggregates. These altered properties were relatively more pronounced in the mutant N101D compared with the mutant N123D. Further, compared with heteroaggregates of WT-alphaA and WT-alphaB, the heteroaggregates containing deamidated subunits of either alphaA- or alphaB-crystallins and their counterpart WT proteins showed higher molecular mass, altered tertiary structures, lower exposed hydrophobic surfaces, and reduced chaperone activity. However, the heteroaggregate containing WT-alphaA and deamidated alphaB subunit showed lower chaperone activity, smaller oligomers, and 3-fold lower subunit exchange rate than heteroaggregate containing deamidated alphaA- and WT-alphaB subunits. Together, the results suggested that (a) both Asn residues (Asn-101 and Asn-123) are required for the structural integrity and chaperone function of alphaA-crystallin and (b) the presence of WT-alphaB in the alpha-crystallin heteroaggregate leads to packing-induced structural changes which influences the oligomerization and modulate chaperone activity.     

Cell kinetic status of mouse lens epithelial cells lacking αA- and αB-crystallin

alphaA- and alphaB-crystallins are small heat shock proteins and molecular chaperones that are known to prevent non-specific aggregation of denaturing proteins. Recent work indicates that alphaA-/- lens epithelial cells grow at a slower rate than wild-type cells, and cultured alphaB-/- cells demonstrate increased hyperproliferation and genomic instability, suggesting that these proteins may exert a direct effect on the cell cycle kinetics, and influence cell proliferation. However, the cell cycle parameters of alphaA/alphaBKO (double knockout) cells have not been analyzed. Here we investigate the cell cycle kinetics of synchronized mouse lens epithelial cultures derived from wild-type and alphaA/alphaB double knockout (alphaA/alphaBKO) mice using BrdU labeling of proliferating cells, and flow cytometric analysis. We also provide data on the changing pattern of expression of HSP25, a small heat shock protein in alphaA/alphaBKO and wild-type cells during the cell cycle. Using serum starvation to synchronize cells in the quiescent G0 phase, and restimulation with serum followed by BrdU labeling and flow cytometry, the data indicated that as compared to wild-type cells, a <50% smaller fraction of the alphaA/alphaBKO cells entered the DNA synthetic S phase of the cell cycle. Furthermore, there was a delay in cell cycle transit through S phase in alphaA/alphaBKO cells, suggesting that although capable of entering S phase, the alphaA/alphaBKO cells are blocked in G1 phase, and are delayed in their cell cycle progression. Immunoblot analysis with antibodies to the small heat shock protein HSP25 indicated that although HSP25 increased in G1 phase of wild-type cells, and remained elevated on further progression through the cell cycle, HSP25 accumulation was delayed to S phase in alphaA/alphaBKO cells. These data can be interpreted to indicate that mouse lens epithelial cell progression through the cell cycle is significantly affected by expression of alphaA and alphaB-crystallin.     

Recognition sequence 2 (residues 60-71) plays a role in oligomerization and exchange dynamics of alphaB-crystallin

Previously, using the peptide scan method, we have determined that residues 42-57 and 60-71 in alphaB-crystallin (TSLSPFYLRPPSFLRA, named recognition sequence 1 or RS-1, and WFDTGLSEMRLE, named recognition sequence 2 or RS-2) are involved in interaction with alphaA-crystallin. To understand the significance of the RS-2 region in interactions between alphaA- and alphaB-crystallins, W60R, F61N, and S66G mutants of alphaB-crystallin were made and tested for their ability to interact with alphaA-crystallin. W60R and S66G mutations increased the oligomeric size of alphaB-crystallin by 1.6- and 2.7-fold respectively, whereas the F61N mutation had no effect. The tryptophan fluorescence intensity of alphaBS66G was 1.5-fold higher than that for the wild type. The intrinsic fluorescence of alphaBF61N was marginally lower than that of alphaB, whereas the fluorescence intensity of alphaBW60R decreased by 40% compared with that of alphaB. The relative availability of hydrophobic sites in the mutants was in the following order: alphaBS66G > alphaB = alphaBF61N = alphaBW60R. The far-UV CD profiles for the wild type and alphaB-crystallin mutants indicated no significant changes in their secondary structures, except for alphaBS66G, which showed an increase in alpha-helical content. The near-UV CD profiles of alphaBW60R and alphaBF61N were nearly similar to that of wild type alphaB. On the other hand, alphaBS66G beyond 270 nm exhibited a signature completely different from that of wild type alphaB. Mutations did not alter the chaperone-like activity of these proteins. The W60R mutation did not affect the rate of subunit exchange between alphaB- and alphaA-crystallins. On the other hand, the S66G mutation increased the subunit exchange rate by 100%, whereas the F61N mutation decreased the rate of subunit exchange between alphaB- and alphaA-crystallins by 36%. Our results establish the importance of residues 60-71 in oligomerization of alphaB-crystallin and subunit interaction between alphaB- and alphaA-crystallins.     

Alpha-crystallin regions affected by adenosine 5'-triphosphate identified by hydrogen-deuterium exchange

ATP interaction with lens alpha-crystallins leading to enhanced chaperone activity is not yet well understood. One model for chaperone activity of small heat shock proteins proposes that ATP causes small heat shock proteins to release substrates, which are then renatured by other larger heat shock proteins. A similar role has been proposed for ATP in alpha-crystallin chaperone activity. To evaluate this model, ATP-induced structural changes of native human alpha-crystallin assemblies were determined by hydrogen-deuterium exchange. In these experiments, hydrogen-deuterium exchange, measured by mass spectrometry, gave direct evidence that ATP decreases the accessibility of amide hydrogens in multiple regions of both alphaA and alphaB. The surface encompassed by these regions is much larger than would be shielded by a single ATP, implying that multiple ATP molecules bind to each subunit and/or ATP causes a more compact alpha-crystallin structure. Such a conformational change could release a bound substrate. The regions most affected by ATP are near putative substrate binding regions of alphaA and alphaB and in the C-terminal extension of alphaB. The widespread decrease in hydrogen-deuterium exchange with particularly large decreases near substrate binding regions suggests that ATP releases substrates via both direct displacement and a global conformational change.     

alpha-Crystallin localizes to the leading edges of migrating lens epithelial cells

alpha-crystallin (alphaA and alphaB) is a major lens protein, which belongs to the small heat-shock family of proteins and binds to various cytoskeletal proteins including actin, vimentin and desmin. In this study, we investigated the cellular localization of alphaA and alphaB-crystallins in migrating epithelial cells isolated from porcine lens. Immunofluorescence localization and confocal imaging of alphaB-crystallin in confluent and in migrating subconfluent cell cultures revealed a distinct pattern of subcellular distribution. While alphaB-crystallin localization was predominantly cytoplasmic in confluent cultures, it was strongly localized to the leading edges of cell membrane or the lamellipodia in migrating cells. In accordance with this pattern, we found abundant levels of alphaB-crystallin in membrane fractions compared to cytosolic and nuclear fractions in migrating lens epithelial cells. alphaA-crystallin, which has 60% sequence identity to alphaB-crystallin, also exhibited a distribution profile localizing to the leading edge of the cell membrane in migrating lens epithelial cells. Localization of alphaB-crystallin to the lamellipodia appears to be dependent on phosphorylation of residue serine-59. An inhibitor of p38 MAP kinase (SB202190), but not the ERK kinase inhibitor PD98059, was found to diminish localization of alphaB-crystallin to the lamellipodia, and this effect was found to be associated with reduced levels of Serine-59 phosphorylated alphaB-crystallin in SB202190-treated migrating lens epithelial cells. alphaB-crystallin localization to the lamellipodia was also altered by the treatment with RGD (Arg-Ala-Asp) peptide, dominant negative N17 Rac1 GTPase, cytochalasin D and Src kinase inhibitor (PP2), but not by the Rho kinase inhibitor Y-27632 or the myosin II inhibitor, blebbistatin. Additionally, in migrating lens epithelial cells, alphaB-crystallin exhibited a clear co-localization with the actin meshwork, beta-catenin, WAVE-1, a promoter of actin nucleation, Abi-2, a component of WAVE-1 protein complex and Arp3, a protein of the actin nucleation complex, suggesting potential interactions between alphaB-crystallin and regulatory proteins involved in actin dynamics and cell adhesion. This is the first report demonstrating specific localization of alphaA and alphaB-crystallins to the lamellipodia in migrating lens epithelial cells and our findings indicate a potential role for alpha-crystallin in actin dynamics during cell migration.     

Structural and functional roles of deamidation and/or truncation of N- or C-termini in human alpha A-crystallin

The purpose of the study was to compare the effects of deamidation alone, truncation alone, or both truncation and deamidation on structural and functional properties of human lens alphaA-crystallin. Specifically, the study investigated whether deamidation of one or two sites in alphaA-crystallin (i.e., alphaA-N101D, alphaA-N123D, alphaA-N101/123D) and/or truncation of the N-terminal domain (residues 1-63) or C-terminal extension (residues 140-173) affected the structural and functional properties relative to wild-type (WT) alphaA. Human WT-alphaA and human deamidated alphaA (alphaA-N101D, alphaA-N123D, alphaA-N101/123D) were used as templates to generate the following eight N-terminal domain (residues 1-63) deleted or C-terminal extension (residues 140-173) deleted alphaA mutants and deamidated plus N-terminal domain or C-terminal extension deleted mutants: (i) alphaA-NT (NT, N-terminal domain deleted), (ii) alphaA-N101D-NT, (iii) alphaA-N123D-NT, (iv) alphaA-N101/123D-NT, (v) alphaA-CT (CT, C-terminal extension deleted), (vi) alphaA-N101D-CT, (vii) alphaA-N123D-CT, and (viii) alphaA-N101/123D-CT. All of the proteins were purified and their structural and functional (chaperone activity) properties determined. The desired deletions in the alphaA-crystallin mutants were confirmed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometric analysis. Relative to WT-alphaA homomers, the mutant proteins exhibited major structural and functional changes. The maximum decrease in chaperone activity in homomers occurred on deamidation of N123 residue, but it was substantially restored after N- or C-terminal truncations in this mutant protein. Far-UV circular dichroism (CD) spectral analyses generally showed an increase in the beta-contents in alphaA mutants with deletions of N-terminal domain or C-terminal extension and also with deamidation plus above N- or C-terminal deletions. Intrinsic tryptophan (Trp) and total fluorescence spectral studies suggested altered microenvironments in the alphaA mutant proteins. Similarly, the ANS (8-anilino-1-naphthalenesulfate) binding showed generally increased fluorescence with blue shift on deletion of the N-terminal domain in the deamidated mutant proteins, but opposite effects were observed on deletion of the C-terminal extension. Molecular mass, polydispersity of homomers, and the rate of subunit exchange with WT-alphaB-crystallin increased on deletion of the C-terminal extension in the deamidated alphaA mutants, but on N-terminal domain deletion these values showed variable results based on the deamidation site. In summary, the data suggested that the deamidation alone showed greater effect on chaperone activity than the deletion of N-terminal domain or C-terminal extension of alphaA-crystallin. The N123 residue of alphaA-crystallin plays a crucial role in maintaining its chaperone function. However, both the N-terminal domain and C-terminal extension are also important for the chaperone activity of alphaA-crystallin because the activity was partially or fully recovered following either deletion in the alphaA-N123D mutant. The results of subunit exchange rates among alphaA mutants and WT-alphaB suggested that such exchange is an important determinant in maintenance of chaperone activity following deamidation and/or deletion of the N-terminal domain or C-terminal extension in alphaA-crystallin.     

Subunit exchange of polydisperse proteins: mass spectrometry reveals consequences of alphaA-crystallin truncation

The small heat shock protein, alpha-crystallin, plays a key role in maintaining lens transparency by chaperoning structurally compromised proteins. This is of particular importance in the human lens, where proteins are exposed to post-translational modifications over the life-time of an individual. Here, we examine the structural and functional consequences of one particular modification of alphaA-crystallin involving the truncation of 5 C-terminal residues (alphaA(1-168)). Using novel mass spectrometry approaches and established biophysical techniques, we show that alphaA(1-168) forms oligomeric assemblies with a lower average molecular mass than wild-type alphaA-crystallin (alphaA(WT)). Also apparent from the mass spectra of both alphaA(WT) and alphaA(1-168) assemblies is the predominance of oligomers containing even numbers of subunits; interestingly, this preference is more marked for alphaA(1-168). To examine the rate of exchange of subunits between assemblies, we mixed alphaB crystallin with either alphaA(WT) or alphaA(1-168) and monitored in a real-time mass spectrometry experiment the formation of heteroligomers. The results show that there is a significant decrease in the rate of exchange when alphaA(1-168) is involved. These reduced exchange kinetics, however, have no effect upon chaperone efficiency, which is found to be closely similar for both alphaA(WT) and alphaA(1-168). Overall, therefore, our results allow us to conclude that, in contrast to mechanisms established for analogous proteins from plants, yeast, and bacteria, the rate of subunit exchange is not the critical parameter in determining efficient chaperone behavior for mammalian alphaA-crystallin.     

An atypical form of alphaB-crystallin is present in high concentration in some human cataractous lenses. Identification and characterization of aberrant N- and C-terminal processing.

Two unique polypeptides, 22.4 and 16.4 kDa, were prominent in some human cataracts. Both proteins were identified as modified forms of the small heat shock protein, alphaB-crystallin. The concentration of total alphaB-crystallin in most of these cataracts was significantly increased. The 22.4-kDa protein was subsequently designated as alphaB(g). Mass spectrometric analyses of tryptic and Asp-N digests showed alphaB(g) is alphaB-crystallin minus the C-terminal lysine. alphaB(g) constituted 10-90% of the total alphaB-crystallin in these cataracts and was preferentially phosphorylated over the typical form of alphaB-crystallin. Human alphaB(g) and alphaB-crystallin were cloned and expressed in Escherichia coli. The differences in electrophoretic mobility and the large difference in native pI values suggest some structural differences exist. The chaperone-like activity of recombinant human alphaB(g) was comparable to that of recombinant human alphaB-crystallin in preventing the aggregation of lactalbumin induced by dithiothreitol. The mechanism involved in generating alphaB(g) is not known, but a premature termination of the alphaB-crystallin gene was ruled out by sequencing the polymerase chain reaction products of the last exon for the alphaB-crystallin gene from lenses containing alphaB(g). The 16.4-kDa protein was an N-terminally truncated fragment of alphaB(g). The high concentration of alphaB-crystallin in these cataracts is the first observation of this kind in human lenses.