Destabilization of lens protein conformation by glutathione mixed disulfide

Mixed disulfide between lens crystallin and glutathione has been observed in human cataracts and could be formed in vitro by thiol-disulfide exchange reaction. The glutathionyl crystallins have been reported to become partially unfolded. The present paper reports the conformational destabilization by the mixed disulfide formation in calf alpha- and gamma-II crystallin. The conformational stability was studied by the denaturants urea and guanidine hydrochloride (Gdn-HCl), and by proteolytic degradation. The denaturation curves of both urea and Gdn-HCl shift to lower denaturant concentration for crystallins of glutathione mixed disulfide. The decrease in conformational stability is estimated to be 0.22- and 0.92 kcal mol-1 for modified alpha- and gamma-II crystallin, respectively. Proteolytic digestion also shows a faster rate of degradation for the modified crystallins. These results indicate that mixed disulfide destabilizes the crystallin conformation. The destabilization may make crystallins more susceptible to changes as observed in aging lenses.     

Effect of oxidized betaB3-crystallin peptide on lens betaL-crystallin: interaction with betaB2-crystallin

PURPOSE: To investigate the interaction of oxidized betaB3-crystallin peptide (residues 152-166) with betaL-crystallin and to identify peptide-interaction sites. METHODS: Peptides were oxidized by using CuSO4 and H2O2. Aggregation and light-scattering assays of bovine betaL-crystallin were conducted at 55 degrees C and 37 degrees C, respectively. Assays were performed in the presence of oxidized and nonoxidized betaB3-crystallin peptides and in the presence of alpha-crystallin. Peptide-induced change in hydrophobicity was determined by bis-ANS (4,4'-dianilino-1,1' binaphthyl-5,5' disulfonic acid) binding study. Oxidized betaB3-peptide binding sites were identified by sulfo-SBED (sulfosuccinimidyl-2-[6-(biotinamido)-2-{p-azidobenzamido}-hexanoamido] ethyl-1-3 dithiopropionate) labeling and mass spectrometric analysis. RESULTS: Aggregation and relative light-scattering of betaL-crystallin was higher in the presence of oxidized betaB3-crystallin peptide than with betaL-crystallin, without oxidized peptide and with nonoxidized peptide. Enhanced aggregation was observed despite the presence of alpha-crystallin in the assay. Furthermore, a significant increase in aggregation and light-scattering was observed in the presence of oxidized betaB3-peptide at 37 degrees C. Bis-ANS binding to betaL-crystallin treated with oxidized betaB3-peptide was two to three times higher than in the controls at 37 degrees C. The oxidized betaB3-peptide preferentially interacted with betaB2-crystallin. The data were confirmed by mass spectrometric analysis. CONCLUSIONS: Oxidized betaB3-peptide interacts with betaB2-crystallin and enhances its aggregation and precipitation. Peptide-induced aggregation and increased hydrophobicity of the lens crystallin at 37 degrees C are relevant to crystallin aggregation in the aging lenses.     

Proteolysis by m-calpain enhances in vitro light scattering by crystallins from human and bovine lenses

PURPOSE: To determine if proteolysis by the calcium-activated protease m-calpain (EC 34.22.17) enhances in vitro light scattering in crystallins from human and bovine lenses. METHODS: Total soluble proteins from bovine, human, and rodent lenses, betaH crystallin, or recombinant betaB1 polypeptide were pre-incubated in the presence or absence of activated m-calpain. Heat-induced light scattering was assayed by measuring changes in optical density at 405 nm. Proteolysis and cleavage sites were detected by SDS-PAGE, two dimensional electrophoresis, and N-terminal Edman sequencing. RESULTS: The in vitro cleavage sites produced by m-calpain on the N-termini of human betaB1, betaA3, and betaB2-crystallins were similar to some of those on bovine and rat crystallins. Proteolysis of alpha- and beta-crystallins was associated with enhanced, heat-induced light scattering by human and bovine lens proteins. CONCLUSIONS: Proteolysis may be a contributing factor in the insolubilization of crystallins occurring during normal maturation of lens or during cataract formation in such species as man and cows.     

Changes in crystallin concentration in rat aqueous and vitreous humors after selenium-induced reversible cortical cataract

Five months after selenite injection, 58% of the rats that had developed cataract earlier underwent a reversal of the cortical opacity. The purpose of this study was to determine if lenses undergoing recovery from cortical opacity reestablish their ability to retain crystallins. By direct ELISA method, the aqueous humor (AH) of control rats was found to contain 18, 39 and 10 ng/ml alpha-, beta- and gamma-crystallin, respectively, while vitreous humor (VH) contained 43, 98 and 23 ng/ml of alpha-, beta- and gamma-crystallin, respectively. In rats with mature cataracts which did not recover by 5 months after selenite injection, there was an approximately 10-fold greater crystallin concentration in the AH and about 20 times greater crystallin concentration in the VH than in the controls. In contrast, rats undergoing recovery from cortical cataract showed almost normal concentration of crystallins in the AH. While crystallins were still elevated in the VH of the rats undergoing recovery from cortical cataract, the crystallin content was lower than in the rats which did not recover. Higher crystallin concentrations in the VH could be explained by either a greater loss through the posterior capsule, or a slower turnover of VH. Decreased crystallin loss from lenses undergoing recovery suggested that the recovered lens at least partly reestablishes its ability to retain crystallins. These data may demonstrate that the lens fiber permeability is lowered while lens repair is occurring.     

Biochemical characterization of lens crystallins from three mammalian species

Lens crystallins were isolated from the homogenates of mammalian eye lenses derived from three different species by gel permeation chromatography and characterized by SDS-gel electrophoresis, isoelectric focusing, amino acid analysis and N-terminal sequence analysis. Five fractions corresponding to HM alpha-, alpha-, beta H-, beta L- and gamma-crystallins were obtained for the crystallins from these phylogenetically distant species. The native molecular masses for these purified fractions and their polypeptide compositions were determined by gel filtration and SDS-gel electrophoresis respectively, revealing the typical subunit compositions for each classified crystallin. The gel pattern of gamma-crystallins from the marmot lens appeared to be more complex than those of gibbon and deer lenses. Comparison of the amino acid contents of each orthologous class of mammalian crystallins with those of evolutionarily distant species still exhibited similarity in their amino acid compositions. The charge heterogeneity of each crystallin fraction can be detected by isoelectric focusing under denaturing conditions. N-terminal sequence analysis of the crystallin fractions revealed that all fractions except that of gamma-crystallin are N-terminally blocked. Extensive sequence similarity between mammalian gamma-crystallin polypeptides were found, which suggested the close relatedness of gamma-crystallins amongst different species of mammals and also established the heterogeneous nature of this multigene family.     

Fluorescence polarization studies of fluorescein isothiocyanate conjugates of bovine lens crystallins

Bovine lens alpha-, beta H- and gamma-crystallin were labeled with the amine-specific fluorescent probe, fluorescein isothiocyanate (FITC) and studied with steady-state polarization measurements. Rotational relaxation times (rho) were estimated for various crystallins and were compared with calculated values. The observed rho value is considerably faster for alpha- and beta H-crystallin conjugate than the calculated value, indicating existence of a segmental motion of the probe on these two crystallins. The segmental flexibility may result from a less tightly folded structure in these crystallins. alpha-Crystallin isolated from the cow lens nucleus shows a smaller rho value than the young cortical alpha-crystallin. The protein partial unfolding process appears to be age-related, and a possible consequence is that crystallin becomes more susceptible to chemical modifications.     

Crystallins and their synthesis in human lens epithelial cells in tissue culture

Explants of epithelial cells from young human lenses of 5-12 months of age, obtained from patients who underwent surgery for retinopathy of prematurity, were cultured in Dulbecco's modified Eagle's medium supplemented with 20% fetal calf serum. Without exception, every piece of the anterior capsule explant showed cell outgrowth within 48-72 h and resulted in confluent monolayer culture within 2 weeks. From these monolayer cultures, two to three passages of subcultures were obtained by routinely seeding cells in a ratio of 1:4. The doubling times for these human lens epithelium (HLE) cultures during the first 4 weeks of two passages were found to be 24-36 h. In a majority of cultures through the first three passages, more than 12 population doublings were attained. However, no lentoid bodies were formed during this period. These cells were studied for the presence of crystallins and their synthesis. Using SDS-polyacrylamide gel electrophoresis, the presence of alpha- and beta-crystallins was demonstrated in HLE cells through three passages. The amount of alpha-crystallin in the first two passages amounted to nearly 13% of the total protein, but decreased significantly in the third passage. The presence of crystallins was corroborated by antibody reaction to the specific crystallins. Indirect immunofluorescence revealed the presence of actin and vimentin in these cell cultures. The synthesis of crystallins in HLE cultures was shown by the incorporation of [35S]methionine which was time dependent. The crystallin synthesis was found to decrease in third passage when the cell growth slowed down without consistent formation of confluent monolayer. These studies have demonstrated that primary cultures of HLE cells can be successfully grown from young lenses through several passages which continue to express the characteristic crystallins of the epithelial cells.     

Probing the structure and interactions of crystallin proteins by NMR spectroscopy

The lens is composed primarily of proteins, the crystallins, at high concentration whose structure and interactions are responsible for lens transparency. As there is no protein turnover in the majority of the lens, crystallin proteins have to be very stable and long-lived proteins. There are three types of crystallin proteins: alpha, beta and gamma, and they all are composed of a variety of subunits. In addition, extensive post-translational modification is undergone by many of the subunits. Determining the structural features and the preferential interactions and associations undergone by the crystallin proteins in the lens is a large and complex experimental undertaking. Some progress has been made in this area by X-ray crystallographic determination of structures for representative examples of the beta- and gamma-crystallins [Slingsby, C., Norledge, B., Simpson, A., Bateman, O. A., Wright, G., Driessen H. P. C., Lindley, P. F., Moss, D. S. and Bax, B. (1997) X-ray diffraction and structure of crystallins. Prog. Ret. Eye Res. 16, 3-29]. In this article, a summary is given of nuclear magnetic resonance (NMR) methods to determine information about these aspects of crystallin proteins. It is shown that despite their relatively large size, all crystallins give rise to well-resolved NMR spectra which arise from flexible terminal extensions that extend from the domain core of the proteins. By examining NMR spectra of mixtures of different crystallin subunits, it is possible to determine the role of these extensions in crystallin-crystallin interactions. For example, the flexible C-terminal extensions in the two alpha-crystallin subunits are not involved in interacting with the other crystallins but are crucially important in the chaperone action of alpha-crystallin. In this action, alpha-crystallin stabilises other proteins under conditions of stress, e.g. heat. In the lens, this ability probably has important consequences in preventing the precipitation of crystallin proteins with age and thereby contributing to cataract formation. The C-terminal extensions in alpha-crystallin act as solubilising agents for the protein and the high-molecular-weight complex that forms upon chaperone action with a precipitating "substrate" protein. Similar behaviour is observed for a variety of small heat-shock proteins, to which alpha-crystallin is related. NMR studies are also consistent with a two-domain structure for alpha-crystallin. No crystal structure is available for crystallin. Using the NMR data, a model for the quaternary structure of alpha-crystallin is proposed which comprises an annular arrangement for the subunits with a large central cavity.     

Monoclonal antibodies to chick crystallins

Several monoclonal antibodies against chick crystallins were obtained. Immunoblot analysis indicated that the monoclonal antibodies distinguish not only the three classes of crystallin (alpha, beta and delta), but also their subclasses. Monoclonal antibodies alpha 2 and alpha 1 reacted exclusively with alpha A and alpha B, respectively, demonstrating that alpha-crystallin subclasses of the chicken are antigenically distinct. An immunohistological study utilizing these monoclonal antibodies showed that the two alpha-crystallin subclasses, alpha A and alpha B, are co-expressed in the same cells and are more concentrated in the epithelium than the fibers in 14-day-old chick embryo lenses. However, immunofluorescence suggested different distribution of alpha-crystallin subclasses within an epithelial cell. alpha A is distributed evenly in the cytoplasm, but alpha B is more concentrated in the fiber-proximal side. Using anti-beta monoclonal antibodies, it was shown that beta-crystallins are divided into three distinct subclasses according to their antigenicity: 27-kDa and 25-kDa beta-crystallins are recognized by beta 1 antibody, 26-kDa and 19-kDa beta-crystallins by beta 2 antibody, and 35-kDa beta-crystallins by beta 3 antibody. All of the anti-delta-crystallin monoclonal antibodies (delta 1 to delta 3) obtained here bound to all delta-crystallin molecular species separable by isoelectrofocusing.     

The ability of lens alpha crystallin to protect against heat-induced aggregation is age-dependent.

Alpha crystallin was prepared from newborn and aged bovine lenses. SDS-PAGE and tryptic peptide mapping demonstrated that both preparations contained only the alpha-A and alpha-B chains, with no significant contamination of other crystallins. Compared with alpha crystallin from the aged lens, alpha crystallin from the newborn lens was much more effective in the inhibition of beta L crystallin denaturation and precipitation induced in vitro by heat. Together, these results demonstrate that during the aging process, the alpha crystallins lose their ability to protect against protein denaturation, consistent with the hypothesis that the alpha crystallins play an important role in the maintenance of protein native structure in the intact lens.     

Water-soluble and insoluble crystallins of the developing human fetal lens, analyzed by agarose/polyacrylamide thin-layer isoelectric focusing

Eight human fetal lenses, selected on basis of normality, of a gestational age of 119 to 231 days were analyzed by thin-layer isoelectric focusing (IEF) in agarose/polyacrylamide gels. This method was adapted for the separation of lens crystallins into HM-, alpha-, beta- and gamma-crystallins. It is especially suitable for analysis under non-denaturing conditions of high-molecular-weight crystallins and of insoluble crystallins (WI) solubilized in formamide. The latter could be separated into HM-, alpha- and gamma-crystallins. During fetal development, a considerable increase of gamma-crystallin proportion was observed due to new synthesis. This increase was balanced by a decrease of alpha-crystallin proportion.     

Changes of lens crystallins photosensitized with tryptophan metabolites

The effect of UV irradiation on bovine lens soluble proteins (crystallins) in the presence of tryptophan metabolites was investigated in vitro. The cross-linking of crystallins by UV irradiation was accelerated by kynurenine, 3-hydroxykynurenine, anthranilic acid and 3-hydroxyanthranilic acid. On denaturation of crystallins by photooxidation, alpha-crystallin was characterized by the formation of water-soluble HMW (high molecular weight) protein, while water-insoluble HMW protein was produced from beta- and gamma-crystallin. These HMW aggregates showed cross-linking by non-disulfide covalent bonds. LMW (low molecular weight) peptides were formed by degradation of alpha- and beta-crystallin.     

Transdifferentiated embryonic neuroretina cells: an in vitro system to study crystallin aggregation process

Transdifferentiated embryonic quail neuroretina cells synthesize in vitro crystallins (the lens-specific proteins) and form lentoid bodies (structures that mimic lens fiber cells) which also contain crystallins. A comparative study on the size of crystallins is reported in 7-day-old embryonic quail lenses, in 7-day-old embryonic quail transdifferentiated neuroretina cells (normal and MH2 transformed), and in isolated lentoid bodies. Analyses are performed using Superose FPLC in combination with SDS-PAGE and Western blot procedures. In quail lenses, an apparent 560-580-kDa alpha crystallin homopolymer is found and delta crystallin, the major avian lens protein, is detected as a 180-kDa tetramer. beta Crystallins, present in low amount within the 180-kDa peak, are a heterogeneous population composed of subunits of molecular weight identical to those found in chick lenses. In addition, an apparent 46-kDa monomeric delta crystallin is found. Normal and MH2-transformed neuroretina cultures produce an alpha crystallin polymer of lower molecular weight (450 kDa) and delta crystallin in a monomeric or dimeric form. The Western blot pattern of beta crystallins from MH2-transformed neuroretina cultures is strictly identical to that of quail lens beta crystallins. In particular, the beta B1 crystallin, which is specific to lens fiber cell differentiation, and the major beta 25-kDa crystallin are present. However, analysis of isolated lentoid bodies from normal transdifferentiated quail neuroretina cultures showed alpha and delta crystallins of comparable size to those found in lens extract, in particular the delta crystallin in tetrameric form. The lentoid body lens-like structure could favour the crystallin aggregation process.     

Characterization of disulfide-linked crystallins associated with human cataractous lens membranes

In order to characterize possible disulfide-linked interactions between lens fiber cell membranes and crystallins, two-dimensional diagonal electrophoresis has been used in combination with Western blot analysis. When these blots were probed with monospecific antisera against alpha, beta and gamma crystallins, membrane from five individual normal lenses showed no disulfide-bonded components. Membrane from 13 individual cataractous human lenses showed no disulfide-bonded alpha crystallin, but did show significant amounts of disulfide-bonded beta crystallin in four out of the 13 lenses studied, and significant amounts of disulfide-bonded gamma crystallin in 10 out of the 13 lenses studied. Together, these studies demonstrate that intermolecular disulfide bonding of crystallins to purified fiber cell membranes is found only in cataractous lenses, and that the predominant polypeptide species involved in this interaction is gamma crystallin.     

Alterations of lens protein synthesis in galactosemic rats.

A differential effect on protein synthesis has been demonstrated in the lenses of galactosemic rats. During galactose cataract development the synthesis of lens crystallins is depressed, whereas that of noncrystallin proteins is unaffected. This effect correlates with the influx of Na+ and loss of K+ from the lens. Removal of the galactose diet results in a gradual recovering of crystallin synthesis to normal levels. In vitro the nuclear cataractous lenses leak crystallins into the media; however, upon 5 day's recovery no leak-out of crystallins could be detected. Both decreased synthesis and leak-out probably account for the marked loss of dry weight of cataractous lenses. These results support the hypothesis that crystallin synthesis may be affected by cation imbalance or changes concomitant with such an imbalance.     

Ontogeny and localization of the alpha-, beta-, and delta- crystallin antigens during lens development in mallard embryos

The ontogeny of alpha-, beta-, and delta-crystallin antigens in the developing lens of mallard (Anas platyrhynchos) was investigated by indirect immunofluorescence using specific antibodies to mallard alpha-, beta-, and delta- crystallins raised in rabbits. The IgG fraction from each antiserum was isolated by affinity chromatography usign Protein A Sepharose CL-4B. Fluoresceinee (FITC) or rhodamine (TRITC) conjugated goat anti rabbit (GAR) gamma-globulin was used as the secondary antibody. The results show that alpha-, beta-, and delta-crystallins appear simultaneously in the developing mallard lens and are detectable from 66 hr (stage 15/16). This situation is different from the chick where delta- is known to appear first followed by beta-, and alpha-. This could be due to species variation. In the epithelium, however, as in the chick, delta- emerges first followed by beta-, and alpha- but rather late when compared with the chick and this seems probably due to a difference in the incubation time between the two species. Results from immunofluorescence and Tris-SDS gel electrophoresis demonstrate that alpha A subunit of the alpha-crystallin appears much earlier than the alpha B subunit. We also found that the newly discovered epsilon-crystallin (18) appears long after the three major crystallin classes.     

Interaction of lens alpha and gamma crystallins during aging of the bovine lens

Heterologous, noncovalent interactions of lens crystallins, such as between alpha and gamma crystallin, are thought to play a key role in the transparent properties of the lens. To determine possible interactions between these two types of crystallins, bovine gamma B crystallin in its native state was purified from whole fetal lenses or from the nucleus of aged bovine lenses, and the purified protein was passed over immobilized alpha crystallin, using a surface plasmon resonance instrument (BIAcore 3000) to obtain refractive units (RU) of gamma B binding at equilibrium. The results demonstrate low binding of gamma B crystallin purified from fetal lenses, but higher binding of the same gamma species purified from aged lenses. Together, these results demonstrate that under equilibrium conditions, gamma B crystallin from the aging bovine lens shows increased noncovalent associations with alpha crystallins, consistent with the possibility that such interactions play an important role in the transparent properties of the aged lens.     

Resistance of human betaB2-crystallin to in vivo modification.

Post-translational modifications and/or structural changes induced by modifications are likely causes of the decrease in crystallin solubility associated with aging and the development of cataract. Characterization of human lens crystallins by mass spectrometry has demonstrated that betaB2-crystallin undergoes less modification than any of the other crystallins. As the lens ages, betaB2-crystallin retains its hydrophilic N-terminus while the hydrophilic C-termini of alpha-crystallins and large portions of the N-termini of betaA3/A1 and betaB1 are truncated. The hydrophilic terminal regions of crystallins contribute to their solubility. Furthermore, deamidation and disulfide bond formation, other modifications that may affect solubility by altering conformation, are less extensive in betaB2 than in the other crystallins. This resistance to modification results in higher levels of betaB2 compared with the other crystallins in the water-soluble fraction of older lenses. The solubility of betaB2 and its propensity to form non-covalent associations with less soluble beta-crystallins may contribute to the solubility of the other beta-crystallins. A current hypothesis is that the chaperone-like properties of alpha-crystallins contribute to lens crystallin solubility, particularly in younger lenses. In older lenses, where most of the alpha-crystallins have become water-insoluble, betaB2-crystallins may play a dominant role in lens crystallin solubility.     

Isotachophoresis and immunoelectrophoresis of water-soluble and -insoluble crystallins of the ageing bovine lens

Water-soluble (WS) and water-insoluble (WI) crystallins were prepared from calf and bovine lenses. The WS-crystallins were analyzed by capillary isotachophoresis and by immunoelectrophoresis with polyvalent and alpha-, beta-, gamma-crystallin specific antisera. The WI-crystallins were first solubilized in formamide and then analyzed by the same methods. The WS-crystallins, which increase in weight, but decrease in % with increasing age, show an increase of alpha-crystallins from 36 to 43%, an increase of beta-crystallins from 42 to 48% and a decrease of gamma-crystallins from 22 to 9%. In the WI-crystallins, which increase in weight and in % with increasing age, the alpha-crystallins form the main portion with an increase from 54 to 61%. The beta-crystallins in WI remain fairly constant at about 37% and the gamma-crystallins decrease from 8 to 2%. Quantitatively, the alpha-crystallin is the major constituent of the lens. The alpha-crystallin and the beta-crystallin components are increasingly incorporated in WI with increasing age. The gamma-crystallin components are selectively incorporated into WI-protein. Immunologically, alpha-, beta- and gamma-crystallins were detected by the polyvalent and specific antisera in WS- and in WI- crystallins.     

Effect of UV-A light on the chaperone-like properties of young and old lens alpha-crystallin

PURPOSE: To study the damaging effect of UV-A irradiation on the chaperone-like properties of alpha-crystallin and the subsequent recovery process of young and old bovine lenses. METHODS: Young and old bovine lenses were kept in organ culture. After 24 hours of incubation they were irradiated with UV-A at 365 nm, and optical quality measurements were performed during the experiments (192 hours). alpha-Crystallin and alpha1-, alphaA2-, alphaB1-, and alphaB2-crystallin subunits were analyzed, separated by gel filtration and cation exchange chromatography, respectively, after different culture times. Protein patterns were obtained after two-dimensional (2-D) gel electrophoresis. Chaperone-like activity was determined on the basis of insulin B-chain and betaL-crystallin aggregation assays. Aggregation of alpha-crystallin was analyzed, tryptophan fluorescence measurements were performed, and alpha-crystallin mRNA levels were determined. RESULTS: The water-soluble alpha-crystallin obtained from old lenses compared with young lenses after UV irradiation had decreased chaperone activity, a higher molecular weight, and increased loss of tryptophan fluorescence. Moreover, alpha-crystallin mRNA virtually disappeared, whereas extra spots on the 2-D protein pattern appeared, possibly because of deamidation. CONCLUSIONS: alpha-Crystallin obtained from old lenses is more affected by irradiation than alpha-crystallin derived from young lenses. Moreover, it appeared that alphaB-crystallin from UV-treated old lenses compared with control lenses was less susceptible to UV-A than alphaA-crystallin. It may well be that alphaB-crystallin protects alphaA-crystallin in vivo.