Packing-induced conformational and functional changes in the subunits of alpha -crystallin

The heteroaggregate alpha-crystallin and homoaggregates of its subunits, alphaA- and alphaB-crystallins, function like molecular chaperones and prevent the aggregation of several proteins. Although modulation of the chaperone-like activity of alpha-crystallin by both temperature and chaotropic agents has been demonstrated in vitro, the mechanism(s) of its regulation in vivo have not been elucidated. The subunits of alpha-crystallin exchange freely, resulting in its dynamic and variable quaternary structure. Mixed aggregates of the alpha-crystallins and other mammalian small heat shock proteins (sHSPs) have also been observed in vivo. We have investigated the time-dependent structural and functional changes during the course of heteroaggregate formation by the exchange of subunits between homoaggregates of alphaA- and alphaB-crystallins. Native isoelectric focusing was used to follow the time course of subunit exchange. Circular dichroism revealed large tertiary structural alterations in the subunits upon subunit exchange and packing into heteroaggregates, indicating specific homologous and heterologous interactions between the subunits. Subunit exchange also resulted in quaternary structural changes as demonstrated by gel filtration chromatography. Interestingly, we found time-dependent changes in chaperone-like activity against the dithiothreitol-induced aggregation of insulin, which correlated with subunit exchange and the resulting tertiary and quaternary structural changes. Heteroaggregates of varying subunit composition, as observed during eye lens epithelial cell differentiation, generated by subunit exchange displayed differential chaperone-like activity. It was possible to alter chaperone-like activity of preexisting oligomeric sHSPs by alteration of subunit composition by subunit exchange. Our results demonstrate that subunit exchange and the resulting structural and functional changes observed could constitute a mechanism of regulation of chaperone-like activity of alpha-crystallin (and possibly other mammalian sHSPs) in vivo.     

Evidence for specific subunit distribution and interactions in the quaternary structure of α‐crystallin

The quaternary structure of α‐crystallin is dynamic, a property which has thwarted crystallographic efforts towards structural characterization. In this study, we have used collision‐induced dissociation mass spectrometry to examine the architecture of the polydisperse assemblies of α‐crystallin. For total α‐crystallin isolated directly from fetal calf lens using size‐based chromatography, the αB‐crystallin subunit was found to be preferentially dissociated from the oligomers, despite being significantly less abundant overall than the αA‐crystallin subunits. Furthermore, upon mixing molar equivalents of purified αA‐ and αB‐crystallin, the levels of their dissociation were found to decrease and increase, respectively, with time. Interestingly though, dissociation of subunits from the αA‐ and αB‐crystallin homo‐oligomers was comparable, indicating that strength of the αA:αA, and αB:αB subunit interactions are similar. Taken together, these data suggest that the differences in the number of subunit contacts in the mixed assemblies give rise to the disproportionate dissociation of αB‐crystallin subunits. Limited proteolysis mass spectrometry was also used to examine changes in protease accessibility during subunit exchange. The C‐terminus of αA‐crystallin was more susceptible to proteolytic attack in homo‐oligomers than that of αB‐crystallin. As subunit exchange proceeded, proteolysis of the αA‐crystallin C‐terminus increased, indicating that in the hetero‐oligomeric form this tertiary motif is more exposed to solvent. These data were used to propose a refined arrangement for the interactions of the α‐crystallin domains and C‐terminal extensions of subunits within the α‐crystallin assembly. In particular, we propose that the palindromic IPI motif of αB‐crystallin gives rise to two orientations of the C‐terminus. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Calcium-induced decrease of the thermal stability and chaperone activity of alpha-crystallin

Alpha-crystallin, one of the major proteins in the vertebrate eye lens, acts as a molecular chaperone, like the small heat-shock proteins, by protecting other proteins from denaturing under stress or high temperature conditions. alpha-Crystallin aggregation is involved in lens opacification, and high [Ca(2+)] has been associated with cataract formation, suggesting a role for this cation in the pathological process. We have investigated the effect of Ca(2+) on the thermal stability of alpha-crystallin by UV and Fourier-transform infrared (FTIR) spectroscopies. In both cases, a Ca(2+)-induced decrease in the midpoint of the thermal transition is detected. The presence of high [Ca(2+)] results also in a marked decrease of its chaperone activity in an insulin-aggregation assay. Furthermore, high Ca(2+) concentration decreases Cys reactivity towards a sulfhydryl reagent. The results obtained from the spectroscopic analysis, and confirmed by circular dichroism (CD) measurements, indicate that Ca(2+) decreases both secondary and tertiary-quaternary structure stability of alpha-crystallin. This process is accompanied by partial unfolding of the protein and a clear decrease in its chaperone activity. It is concluded that Ca(2+) alters the structural stability of alpha-crystallin, resulting in impaired chaperone function and a lower protective ability towards other lens proteins. Thus, alpha-crystallin aggregation facilitated by Ca(2+) would play a role in the progressive loss of transparency of the eye lens in the cataractogenic process.     

Effect of Trifluoroethanol on the Structural and Functional Properties of α-Crystallin

Alpha crystallin is an eye lens protein with a molecular weight of approximately 800 kDa. It belongs to the class of small heat shock proteins. Besides its structural role, it is known to prevent the aggregation of β- and γ-crystallins and several other proteins under denaturing conditions and is thus believed to play an important role in maintaining lens transparency. In this communication, we have investigated the effect of 2,2,2-trifluoroethanol (TFE) on the structural and functional features of the native α-crystallin and its two constituent subunits. A conformational change occurs from the characteristic β-sheet to the α-helix structure in both native α-crystallin and its subunits with the increase in TFE levels. Among the two subunits, αA-crystallin is relatively stable and upon preincubation prevents the characteristic aggregation of αB-crystallin at 20% and 30% (v/v) TFE. The hydrophobicity and chaperone-like activity of the crystallin subunits decrease on TFE treatment. The ability of αA-crystallin to bind and prevent the aggregation of αB-crystallin, despite a conformational change, could be important in protecting the lens from external stress. The loss in chaperone activity of αA-crystallin exposed to TFE and the inability of peptide chaperone—the functional site of αA-crystallin—to stabilize αB-crystallin at 20–30% TFE suggest that the site(s) involved in subunit interaction and chaperone-like function are quite distinct.     

Heat-induced changes in the conformation of alpha- and beta-crystallins: unique thermal stability of alpha-crystallin

Of the crystallin proteins of the lens, the principal subunit of the beta-crystallin, beta B2 (beta Bp), has been considered to be the only heat-stable protein because it does not precipitate upon heating. In our recent investigations, however, we have found that the alpha-crystallin from bovine lenses is not only heat stable but also does not denature at temperatures up to 100 degrees C. Using circular dichroism and fluorescence to monitor the conformational changes of alpha- and beta B2-crystallins upon heating, we found that alpha-crystallin maintains a high degree of structure, whereas the beta B2-crystallin shows a reversible sigmoidal order-disorder transition at about 58 degrees C.     

Lens proteasome shows enhanced rates of degradation of hydroxyl radical modified alpha-crystallin

Proteasome, a high molecular weight protease complex (HMP, approximately 600 kDa) was isolated from bovine eye lens epithelium tissue. In contrast with prior reports, lens proteasome degraded the major lens protein alpha-crystallin and S-carboxymethylated bovine serum albumin at 37 degrees C, mostly to trichloroacetic acid precipitable polypeptides. The proteasome, thus isolated, was labile at 55 degrees C. As indicated by the ability of p-chloromercuribenzoate and N-ethylmaleimide to block activity, a thiol group is required for activity. Alpha-crystallin was oxidized by exposure to 60Co-irradiation under an atmosphere of N2O (1-50 kilorads). This dose delivered 0.1-5.7 mol of hydroxyl radicals per mol of crystallin. Irradiation resulted in increased heterogeneity, aggregation, and fragmentation of the crystallin preparation. The proteolytic susceptibility of alpha-crystallin to the lens HMP was enhanced by the irradiation in a dose-dependent manner up to 20 kilorads (.OH concentration up to 2.3 mol per mol of alpha-crystallin). When 50 kilorads (5.7 mol .OH per mol of alpha-crystallin) was used, there was extensive aggregation and no enhancement in proteolysis over the unirradiated sample. The data indicate that the lens HMP can degrade mildly photooxidized lens proteins, but proteins which are extensively damaged are not degraded and may accumulate. This may be related to cataract formation.     

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.     

Significance of interactions of low molecular weight crystallin fragments in lens aging and cataract formation

Analysis of aged and cataract lenses shows the presence of increased amounts of crystallin fragments in the high molecular weight aggregates of water-soluble and water-insoluble fractions. However, the significance of accumulation and interaction of low molecular weight crystallin fragments in aging and cataract development is not clearly understood. In this study, 23 low molecular mass (<3.5-kDa) peptides in the urea-soluble fractions of young, aged, and aged cataract human lenses were identified by mass spectroscopy. Two peptides, alphaB-(1-18) (MDIAIHHPWIRRPFFPFH) and betaA3/A1-(59-74) (SD(N)AYHIERLMSFRPIC), present in aged and cataract lens but not young lens, and a third peptide, gammaS-(167-178) (SPAVQSFRRIVE) present in all three lens groups were synthesized to study the effects of interaction of these peptides with intact alpha-, beta-, and gamma-crystallins and alcohol dehydrogenase, a protein used in aggregation studies. Interaction of alphaB-(1-18) and betaA3/A1-(59-74) peptides increased the scattering of light by beta- and gamma-crystallin and alcohol dehydrogenase. The ability of alpha-crystallin subunits to function as molecular chaperones was significantly reduced by interaction with alphaB-(1-18) and betaA3/A1-(59-74) peptides, whereas gammaS peptide had no effect on chaperone-like activity of alpha-crystallin. The betaA3/A1-(59-74 peptide caused a 5.64-fold increase in alphaB-crystallin oligomeric mass and partial precipitation. Replacing hydrophobic residues in alphaB-(1-18) and betaA3/A1-(59-74) peptides abolished their ability to induce crystallin aggregation and light scattering. Our study suggests that interaction of crystallin-derived peptides with intact crystallins could be a key event in age-related protein aggregation in lens and cataractogenesis.     

Cardiac alpha-crystallin

Rat hearts were perfused in the working heart or Langendorff mode and then subjected to total normothermic ischemia. The content of alpha-crystallin in the water soluble protein fraction obtained from these hearts diminished in a time-dependent manner during ischemia. The protein was recovered in the low g pellet of the homogenate. The redistribution was dramatic, selective for alpha-crystallin and irreversible. Large crystallin clumps formed also when exposing the soluble protein fraction of control hearts to slightly acidic pH (6.5-7.0). Electron microscopic analysis showed that aggregation of the globular homo-oligomeric units of crystallin occurred. The aggregates probably represented denatured protein and were similar in appearance to lenticular alpha H-crystallin. In purified form, however, cardiac crystallin particles did not cluster at pH 6.5. Aggregation only occurred in the presence of other protein components (including, probably, cytosolic actin) of the soluble fraction. A direct and selective interaction between actin and cardiac crystallin could be demonstrated using actin-Sepharose affinity chromatography procedures. The results suggest that large aggregates of cardiac crystallin form very early during ischemia, due to acidification of the cytosol. Cardiac crystallin is highly homologous to stress proteins and is localized on the Z-disks, where it plays probably a structural or protective role. Its rapid and complete denaturation could be involved in the genesis of the irreversible structural damages occurring during ischemia.     

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.     

Identification of Subunit-Subunit Interaction Sites in αA-WT Crystallin and Mutant αA-G98R Crystallin Using Isotope-Labeled Cross-Linker and Mass Spectrometry

Cataract is characterized by progressive protein aggregation and loss of vision. α-Crystallins are the major proteins in the lens responsible for maintaining transparency. They exist in the lens as highly polydisperse oligomers with variable numbers of subunits, and mutations in α-crystallin are associated with some forms of cataract in humans. Because the stability of proteins is dependent on optimal subunit interactions, the structural transformations and aggregation of mutant proteins that underlie cataract formation can be understood best by identifying the residue-specific inter- and intra-subunit interactions. Chemical crosslinking combined with mass spectrometry is increasingly used to provide structural insights into intra- and inter-protein interactions. We used isotope-labeled cross-linker in combination with LC-MS/MS to determine the subunit–subunit interaction sites in cataract-causing mutant αA-G98R crystallin. Peptides cross-linked by isotope-labeled (heavy and light forms) cross-linkers appear as doublets in mass spectra, thus facilitating the identification of cross-linker–containing peptides. In this study, we cross-linked wild-type (αA-WT) and mutant (αA-G98R) crystallins using the homobifunctional amine-reactive, isotope-labeled (d₀ and d₄) cross-linker–BS²G (bis[sulfosuccinimidyl]glutarate). Tryptic in-solution digest of cross-linked complexes generates a wide array of peptide mixtures. Cross-linked peptides were enriched using strong cation exchange (SCX) chromatography followed by both MS and MS/MS to identify the cross-linked sites. We identified a distinct intermolecular interaction site between K88 — K99 in the β5 strand of the mutant αA-G98R crystallin that is not found in wild-type αA-crystallin. This interaction could explain the conformational instability and aggregation nature of the mutant protein that results from incorrect folding and assembly.     

The effect of dextran on subunit exchange of the molecular chaperone alphaA-crystallin

Alpha-crystallin, a member of small heat shock protein (sHsp) family, is comprised of alphaA and alphaB subunits and acts as a molecular chaperone by interacting with unfolding proteins to prevent their aggregation. The alphaA-crystallin homopolymer consists of 30-40 subunits that are undergoing dynamic exchange. In vivo, alpha-crystallin elicits its chaperone action in a crowded cellular environment (e.g. in the lens). In vitro, inert molecular crowding agents (e.g. dextran) are often used to mimic crowded conditions. In this study, it was found that alpha-crystallin and alphaA-crystallin are poorer chaperones in the presence of dextran. Using fluorescence resonance energy transfer, it is shown that the alphaA-crystallin subunit exchange rate strongly increases with temperature. Binding of reduced ovotransferrin to alphaA-crystallin markedly decreases the rate of subunit exchange, as does the presence of dextran. In addition, in the presence of dextran the effect of reduced ovotransferrin on decreasing the rate of subunit exchange of alphaA-crystallin is greater than in the absence of dextran. Under the conditions of molecular crowding, the alphaA-crystallin subunit exchange rate is not temperature-dependent. In the absence of dextran, the exchange rate of alphaA-crystallin subunits correlates with its chaperone efficiency, i.e. the chaperone ability of alphaA-crystallin increases with temperature. However in the presence of dextran, the temperature dependence of the chaperone ability of alphaA-crystallin is eliminated.     

The molecular chaperone, alpha-crystallin, inhibits amyloid formation by apolipoprotein C-II.

Under lipid-free conditions, human apolipoprotein C-II (apoC-II) exists in an unfolded conformation that over several days forms amyloid ribbons. We examined the influence of the molecular chaperone, alpha-crystallin, on amyloid formation by apoC-II. Time-dependent changes in apoC-II turbidity (at 0.3 mg/ml) were suppressed potently by substoichiometric subunit concentrations of alpha-crystallin (1-10 microg/ml). alpha-Crystallin also inhibits time-dependent changes in the CD spectra, thioflavin T binding, and sedimentation coefficient of apoC-II. This contrasts with stoichiometric concentrations of alpha-crystallin required to suppress the amorphous aggregation of stressed proteins such as reduced alpha-lactalbumin. Two pieces of evidence suggest that alpha-crystallin directly interacts with amyloidogenic intermediates. First, sedimentation equilibrium and velocity experiments exclude high affinity interactions between alpha-crystallin and unstructured monomeric apoC-II. Second, the addition of alpha-crystallin does not lead to the accumulation of intermediate sized apoC-II species between monomer and large aggregates as indicated by gel filtration and sedimentation velocity experiments, suggesting that alpha-crystallin does not inhibit the relatively rapid fibril elongation upon nucleation. We propose that alpha-crystallin interacts stoichiometrically with partly structured amyloidogenic precursors, inhibiting amyloid formation at nucleation rather than the elongation phase. In doing so, alpha-crystallin forms transient complexes with apoC-II, in contrast to its chaperone behavior with stressed proteins.     

alpha-Crystallin chaperone-like activity and membrane binding in age-related cataracts.

alpha-Crystallin, the major protein component of the vertebrate lens, is thought to play a critical role in the maintenance of transparency through its ability to inhibit stress-induced protein aggregation. However, during aging and cataract formation the amount of membrane-bound alpha-crystallin increases significantly while high molecular weight complexes (HMWCs) comprised of alpha-crystallin and other lens crystallins accumulate. These and other recent data suggest a possible link between cataract formation, the formation of high molecular weight alpha-crystallin aggregates, and the progressive increase in membrane association of alpha-crystallin. To better understand these processes, we characterized the chaperone-like activity (CLA) and subunit exchange of membrane bound alpha-crystallin. In addition, we measured the membrane binding properties of in vitro constituted HMWCs to understand the mechanism by which increased alpha-crystallin is bound to the membrane of old and cataractous lens cells in vivo. Membrane-associated alpha-crystallin complexes have measurably reduced CLA compared to complexes in solution; however, membrane binding does not alter the time required for alpha-crystallin complexes to reach subunit exchange equilibrium. In addition, HMWCs prepared in vitro have a profoundly increased membrane binding capacity as compared to native alpha-crystallin. These results are consistent with a model in which increased membrane binding of alpha-crystallin is an integral step in the pathogenesis of many forms of cataracts.     

Influence of chemical modification of cysteine and histidine side chains upon subunit reassembly of alpha crystallin

Alpha crystallin, the important multimeric structural protein of mammalian eye lens, is an assembly composed of 30 alpha-A and 10 alpha-B subunits. The influence of either partial or complete chemical modification of two important amino acid side chains, cysteine and histidine, upon the integrity of native alpha crystallin assembly and also upon the mode of subunit reassembly has been investigated. It has been found that chemical modification of surface-exposed cysteine and histidine side chains does not affect the subunit-subunit interactions stabilizing the native aggregate. Cysteine modifications, either partial or complete, unlike histidine modifications, do not seem to affect the backbone conformation of the subunits refolded after denaturation. Both cysteine and histidine modifications, however, affect the packing of the refolded structural elements forming the tertiary structure of the subunits and also the mode of oligomeric reorganization. The most striking effect of histidine modification is the considerable increase in size of the aggregates upon reassociation of the modified subunits. The chaperone activity, however, has been found to remain almost unaffected in spite of these chemical modifications.     

Eye lens alphaA- and alphaB-crystallin: complex stability versus chaperone-like activity.

The major lens protein alpha-crystallin is composed of two related types of subunits, alphaA- and alphaB-crystallin, of which the former is essentially lens-restricted, while the latter also occurs in various other tissues. With regard to their respective chaperone capacities, it has been reported that homomultimeric alphaA-crystallin complexes perform better in preventing thermal aggregation of proteins, while alphaB-crystallin complexes protect more efficiently against reduction-induced aggregation of proteins. Here, we demonstrate that this seeming discrepancy is solved when the reduction assay is performed at increasing temperatures: above 50 degrees C alphaA- performs better than alphaB-crystallin also in this assay. This inversion in protective capacity might relate to the greater resistance of alphaA-crystallin to heat denaturation. Infrared spectroscopy, however, revealed that this is not due to a higher thermostability of alphaA-crystallin's secondary structure. Also the accessible hydrophobic surfaces do not account for the chaperoning differences of alphaA- and alphaB-crystallin, since regardless of the experimental temperature alphaB-crystallin displays a higher hydrophobicity. It is argued that the greater complex stability of alphaA-crystallin, as evident upon urea denaturation, and the higher chaperone capacity of alphaB-crystallin at physiological temperatures reflect the evolutionary compromise to obtain an optimal functioning of heteromeric alpha-crystallin as a lens protein.     

Influence of chemical modification of cysteine and histidine side chains upon subunit reassembly of alpha crystallin

Alpha crystallin, the important multimeric structural protein of mammalian eye lens, is an assembly composed of 30 alpha-A and 10 alpha-B subunits. The influence of either partial or complete chemical modification of two important amino acid side chains, cysteine and histidine, upon the integrity of native alpha crystallin assembly and also upon the mode of subunit reassembly has been investigated. It has been found that chemical modification of surface-exposed cysteine and histidine side chains does not affect the subunit-subunit interactions stabilizing the native aggregate. Cysteine modifications, either partial or complete, unlike histidine modifications, do not seem to affect the backbone conformation of the subunits refolded after denaturation. Both cysteine and histidine modifications, however, affect the packing of the refolded structural elements forming the tertiary structure of the subunits and also the mode of oligomeric reorganization. The most striking effect of histidine modification is the considerable increase in size of the aggregates upon reassociation of the modified subunits. The chaperone activity, however, has been found to remain almost unaffected in spite of these chemical modifications.     

Glycation by ascorbic acid oxidation products leads to the aggregation of lens proteins

Previous studies from this laboratory have shown that there are striking similarities between the yellow chromophores, fluorophores and modified amino acids released by proteolytic digestion from calf lens proteins ascorbylated in vitro and their counterparts isolated from aged and cataractous lens proteins. The studies reported in this communication were conducted to further investigate whether ascorbic acid-mediated modification of lens proteins could lead to the formation of lens protein aggregates capable of scattering visible light, similar to the high molecular aggregates found in aged human lenses. Ascorbic acid, but not glucose, fructose, ribose or erythrulose, caused the aggregation of calf lens proteins to proteins ranging from 2.2 x 10(6) up to 3.0 x 10(8 )Da. This compared to proteins ranging from 1.8 x 10(6) up to 3.6 x 10(8 )Da for the water-soluble (WS) proteins isolated from aged human lenses. This aggregation was likely due to the glycation of lens crystallins because [U-(14)C] ascorbate was incorporated into the aggregate fraction and because NaCNBH(3), which reduces the initial Schiff base, prevented any protein aggregation. Reactions of ascorbate with purified crystallin fractions showed little or no aggregation of alpha-crystallin, significant aggregation of beta(H)-crystallin, but rapid precipitation of purified beta(L)- and gamma-crystallin. The aggregation of lens proteins can be prevented by the binding of damaged crystallins to alpha-crystallin due to its chaperone activity. Depending upon the ratios between the components of the incubation mixtures, alpha-crystallin prevented the precipitation of the purified beta(L)- and gamma-crystallin fractions during ascorbylation. The addition of at least 20% of alpha-crystallin by weight into glycation mixtures with beta(L)-, or gamma-crystallins completely inhibited protein precipitation, and increased the amount of the high molecular weight aggregates in solution. Static and dynamic light scattering measurements of the supernatants from the ascorbic acid-modified mixtures of alpha- and beta(L)-, or gamma-crystallins showed similar molar masses (up to 10(8 )Da) and hydrodynamic diameter (up to 80( )nm). These data support the hypothesis, that if the lens reducing environment is compromised, the ascorbylation of lens crystallins can significantly change the short range interactions between different classes of crystallins leading to protein aggregation, light scattering and eventually to senile cataract formation.     

The thermal structural transition of α-crystallin inhibits the heat induced self-aggregation

α-crystallin, the major constituent of human lens, is a member of the heat-shock proteins family and it is known to have a quaternary structural transition at . The presence of calcium ions and/or temperature changes induce supramolecular self-aggregation, a process of relevance in the cataractogenesis. Here we investigate the potential effect of the bovine α-crystallin's structural transition on the self-aggregation process. Along all the temperatures investigated, aggregation proceeds by forming intermediate molecular assemblies that successively aggregate in clusters. The final morphology of the aggregates, above and below Tc, is similar, but the aggregation kinetics are completely different. The size of the intermediate molecular assemblies, and their repulsive energy barrier show a marked increase while crossing . Our results highlight the key role of heat modified form of α-crystallin in protecting from aggregation and preserving the transparency of the lens under hyperthermic conditions.     

An attempt to assay the state of determination by using transfected genes as probes in transdifferentiation of neural retina into lens

Hybrid genes coding for chloramphenicol acetyltransferase (CAT) with a non-specific retroviral, lens-specific delta-crystallin or lens-specific alpha-crystallin promoters were constructed to transfect the transdifferentiating (lentoidogenic) and non-transdifferentiating (non-lentoidogenic) cultures of chicken embryonic neural retina for assaying the state of determination towards lens differentiation. The expression occurred only when CAT genes with lens-specific promoters were transfected to the cultures maintained in the conditions permissive to lentoidogenesis. The expression of these exogenous, lens-specific CAT genes began at stages of culturing that were earlier than the expression of endogenous crystallin. Presumably, there are two steps in the transdifferentiation of neural retina into lens; acquisition of capacity to express crystallin genes and derepression of the endogenous crystallin genes.