Steroid adduct formation with lens crystallins

Background : Development of steroid cataract is a likely outcome following prolonged exposure to glucocorticoids. It has been suggested that formation of steroid-protein adducts is a key event in this lens opacification. In order to explore this possibility, we have monitored the reaction of bovine lens proteins with glucocorticoids and examined the effects of adduct formation on their structures. Methods : Bovine lens proteins were incubated with high (10^?4 M) and low (10^?8 M) concentrations of dexamethasone or prednisolone for up to 56 days at 37 degrees Celsius. Changes in molecular size and solubility of the crystallins and their polypeptide subunits were examined using gel permeation chromatography and SDS gel electrophoresis. Conformational changes were assessed with the aid of tryptophan fluorescence spectroscopy and oxidation was monitored by measuring protein sulphydryl content. Results : Covalent incorporation of glucocorticoids was observed for all crystallins with relative reactivities for α-: β-: γ-crystallin of 20: 5: 1. The maximum incorporated was one steroid molecule per 40 to 50 subunits of α-crystallin. The proportions and sizes of the soluble crystallins and their subunits were unchanged. Protein sulphydryl contents decreased by eight to 10 per cent more than controls but no intermolecular disulphide bonds were detected. There were no alterations in tryptophan microenvironments. Conclusions : Steroids form adducts with lens proteins, in particular α-crystallin, but it appears unlikely that this reaction is responsible for steroid cataract formation.     

Effects of temperature, concentration and carboxymethylation on interactions of calf lens crystallins.

Concentration and temperature dependent interactions of calf cortical crystallins were studied by means of gel filtration and disc gel electrophoretic analyses. Low temperature (8°C) gel filtration of either low or high levels of cortical crystallin extracts (5–59 A₂₈₀ units) yields elution profiles that are indistinguishable in the distributions of α-, β_H-, β_L- and γ-crystallins. Gel filtration of low levels of extracts between 22 and 44°C causes a gradual but ultimately complete temperature-dependent disappearance of the region of β_H-crystallins and to a lesser degree in the γ-crystallin peak. Chromatography of high levels of extracts at 44°C results in the reappearance of a high mol. wt. β-crystallin peak with electrophoretic property and polypeptide composition different from those of β_H-crystallin isolated at the low temperature of 8°C. Preincubation of high levels of extracts at 44°C causes some small but irreversible changes in the elution profile obtained at 8°C. Taken together these results suggest that the presence of high mol. wt. β-crystallin is the result of largely reversible equilibria between β-crystallins and other lenticular components.The reaction of calf cortical extracts with increasing amounts of iodoacetic acid results in a progressive loss in the β_H-crystallin region of the 8°C filtration profile. Results from amino acid analyses and sodium dodecylsulfate (SDS) gel electrophoreses of the respective α-, β_L- and γ-crystallin fractions indicate that the SH moieties of the cysteine residues are essential to the formation of high mol. wt. β-crystallins.     

Immunohistochemical studies of lens crystallins in the dysgenetic lens (dyl) mutant mice

The lens in the dyl mutant mice shows a persistent lens-ectodermal connection as well as degeneration and extrusion of lens materials after the initial differentiation of the fibres. Immunohistochemical investigation of the ontogeny of the lens crystallins in this developing mutant lens has been carried out using the indirect immunofluorescence staining method with antiserum to adult mouse lens total soluble proteins. The results have been compared with those for coisogenic normal lens used as a control. In both, the first positive reaction was detectable at identical stages of lens development. A rapid increase in the intensity of fluorescence, most marked in the elongating fibre progressing through the equatorial region to the epithelium, was recorded in the mutant as well as in the normal lens. However, the stalk leading to the lens epithelium did not show any reaction. Appearance of vacuoles in the lens nucleus and cortex marked the beginning of degeneration of fibres which otherwise showed strong fluorescence. This was followed by extrusion of lens crystallin materials through the stalk. As a result, the lens became increasingly reduced and malformed but the surviving cells making up the vestigeal lens in the adult showed positive immunofluorescence. The results demonstrate that despite a failure of lens-ectoderm separation in the mutant mice, the ontogeny of the lens crystallins and differentiation of the lens up to a certain stage of development follow an apparently normal course before the commencement of cataractous degeneration.     

Radioimmunoassay of sheep alpha- and gamma-crystallins II. Organ specificity of lens crystallins

The α- and γ-crystallin content in sheep ocular tissues was assayed by radioimmunoassay.In the normal sheep eye, the amounts assayed in extralenticular tissue extracts were found to be close to the lower limit of sensitivity of the assays, 2 ng/ml. making the nature of the measured effects uncertain. The concentrations found, indicated a crystallin content of less than 0·05 parts per million wet weight tissue. This concentration is so low that it is considered unlikely that the crystallins are produced outside the lens.Freezing and thawing of the intact eye caused leakage of crystallins from the lens to the surrounding tissues. Because of the enormous concentration gradient of crystallins across the lens capsule, even minute damage to the capsule was shown to cause crystallin contamination of the surrounding tissues. One sheep with Peter's anomaly in one eye had increased amount of crystallins outside the lens in both eyes.     

Specific radioimmunoassays for human alpha and gamma crystallins

The establishment of specific radioimmunoassays for human α- and γ-crystallins is described. The assays were used to evaluate the protein fractions obtained by conventional gel filtration chromatography and ion exchange chromatography of human lenses. It was demonstrated that only young lenses gave homogenous crystallin fractions, the fractions from older lenses consisted of mixtures of various crystallins. With increasing age or cataract formation no increase in α- or decrease in γ-crystallin concentration was found in the soluble protein fractions.     

晶状体蛋白的翻译后修饰

晶状体蛋白（α-、β-、γ-晶状体蛋白）可发生翻译后修饰,且在不同的位点发生不同类型的翻译后修饰,从而影响晶状体蛋白的功能.部分翻译后修饰不仅增加晶状体蛋白的不溶性,也改变晶状体蛋白的构象,最终促进白内障的形成;有些翻译后修饰则与白内障的形成无关,可能与晶状体蛋白的发育或保护作用有关.研究晶状体蛋白的翻译后修饰及调控,对深入了解晶状体发育、衰老、白内障形成等过程有重要意义.就其在蛋白质水平上阐明有关晶状体的生理及病理过程进行综述.     

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.     

A light microscope study of the lens of the tryptophan deficient rat.

The lens of the rat fed on a tryptophan-deficient diet is smaller than normal and many of the cortical fibre cells have abnormal morphologies. These cells are probably related to, if not the direct cause of, the inner reflecting surface seen within the lens when the eye is viewed in the slit lamp.The sequence of changes in crystallin synthesis during the formation of lens fibre cells in the newborn tryptophan-deficient rat was examined by immunofluorescence. There were no differences between the normal and the tryptophan-deficient rat in the distribution of α-, β- and γ-crystallins. Therefore, in this respect at least, the differentiation of lens fibres in newborn tryptophan-deficient animals was normal.     

Radioimmunoassay of sheep alpha- and gamma-crystallins I. 125I labelling of sheep gamma-crystallin. Characterization of the assays in detecting homologous and heterologous lens crystallins

A radioimmunoassay for sheep γ-crystallin is described. ¹²⁵I labelling of native γ-crystallin is difficult to accomplish, probably due to its high thiol content but after alkylation labelling became satisfactory. Gamma crystallin tends to form complexes with α-crystallin, making it difficult to obtain a pure α-crystallin preparation. The described immunoassay is specific for γ-crystallin and is not influenced by the presence of other lens crystallins. The lower limit of sensitivity of the assay is 2 ng/ml. The assay is compared to the previously described assay for α-crystallin. The assays were used to evaluate cross reactions between α- and γ-crystallins from four different species.     

Immunochemical studies on lens protein-protein complexes III. Age-related changes in the stability of lens alpha crystallin containing compleces

The age dependence of the stability of lens crystallin complexes was investigated. The lens protein fractions retained on Diaflo XM-300 membranes were subsequently separated on Bio-gel A 15 m columns, yielding one calf cortical α-β crystallin complex and two different bovine nuclear α-β-γ crystallin complexes. Separate samples of these different lens crystallin complexes were exposed for various lengths of time to acidic or alkaline pH, or to high concentrations of salts at neutral pH. Acidic pH, three units below the neutral, had a more pronounced dissociating effect on the crystallin complexes than alkaline pH, three units above the neutral, or a high salt concentration. The dissociation of the various complexes was tested by ultrafiltration on Diaflo XM-300 membranes and by immunochemical analysis of the fractions retained and filtered.Comparison of the calf cortical and bovine nuclear complex preparations indicated that the stability of the crystallin complexes increases with progressive aging of the lens. The crystallin complexes investigated did dissociate in a constant electrical field, yielding free α-, β- and γ-crystallin molecules.     

Characterization of chick lens soluble proteins and the control of their synthesis

A single step column procedure for the separation of chick lens soluble proteins is described. α-, β- and δ-crystallin, which account for 80–90% of the soluble protein are separated using this technique, with little or no cross-contamination between classes, as judged by immuoelectrophoresis. Analysis of the fractions in sodium dodecylsulphate-polyacrylamide (SDS) and isoelectric focusing (IEF) gels in dissociating conditions identifies and characterizes the major α-, β- and δ-crystallin subunits. Fresh lenses from day-old chicks were labelled by culturing in medium containing radioactive amino acids and the synthesis of individual crystallin subunits analysed. In both pulse and pulse-chase labelled lenses at least 70% of the incorporated radioactivity could be assigned to characterized crystallin subunits. Differences were observed in the radioactivity profiles from pulse and pulse-chase labelled lenses when analysed on SDS and IEF gels and some polypeptides showed labelling characteristics typical of post-translational modifications of existing protein chains.     

The development and application of a radioimmunoassay to lens crystallins

A radioimmunoassay was developed for mouse α- and γ-crystallins. A standard inhibition curve against known quantities of the crystallins served as a basis for quantitating unknown samples. The sensitivity of the assay was 2 ng for α-crystallin and 4 ng for γ-crystallin. It was possible to quantitate the amount of γ-crystallin present in the cultures from normal mouse and Nakano mouse lenses. The results were consistent with the previous immunofluorescent observation of γ-crystallin localization in the lentoid bodies. In addition, changes in the lens crystallins in normal and Nakano mouse lenses were followed. A dramatic decrease of γ-crystallin occurred during cataract development, however the level of α-crystallin in the soluble lens protein remained unchanged during this period.     

Analysis of the proteins in the rhesus monkey lens

The proteins of the Rhesus monkey (Macaca mulatta) lens were studied and characterized. In general, there is a similarity of the monkey lens to the human lens particularly in the alpha- and beta-crystallins and the membrane proteins, although the amount of beta 1-crystallin may be greater in the monkey. There is immunological cross-reactivity in the crystallins between monkey and human lenses including the monkey low molecular weight proteins and the human low molecular weight proteins. The monkey lens low molecular weight material showed three differently sized proteins by gel exclusion chromatography. In this respect, the monkey lens proteins are similar to the human. However, the monkey lens low molecular weight proteins differ from the human low molecular weight proteins in charge as well as molecular weight determined by SDS-PAGE. One of the monkey low molecular weight proteins is more prevalent in the nucleus than in the cortex suggesting that this protein may be more important to the embryonic lens that to the adult lens.     

Immunochemical studies on lens protein-protein complexes I. The heterogeneity and structure of complexed alpha-crystallin

A buffered extract of young cattle lenses filtered through Diaflo XM-300 membranes, retaining molecules above 300 000 daltons, was found to contain in the non-filterable fraction constituting 50% of soluble lens proteins α-and β-crystallins. The non-filterable fraction of soluble proteins from mature cattle lenses, constituting 87% of all crystallins, contained not only α- and β-crystallins, but γ-crystallins as well. Only α-crystallin, with a molecular weight above 300 000 daltons, was expected to remain in the non-filterable fraction. All of the β-crystallins, which have a molecular weight below 200 000 daltons, and γ-crystallins, with a molecular weight of about 20 000 daltons, if free in solution and not complexed, were expected to be in the filtrate.The lens proteins with a molecular weight above 300 000 daltons from young lens tissue were separated by gel chromatography (Bio-Rad A 15 m) into free α-crystallin followed by an α-β-crystallin complex. By the same procedure, the proteins above 300 000 daltons from mature lenses were separated into two complexes of quantitatively different α-β-γ-crystallin composition. A dependence of lens protein complex formation on the ionic composition of the solvent was observed. The differences between calf cortical and bovine nuclear complexes, found in buffer extracts, were much less pronounced in water extracts in which both contained γ-crystallins.Specific dissociation of the lens protein complexes was obtained by use of an anti-α-crystallin immunoadsorbent, as shown by the passage of the previously retained β- and γ-crystallins through a Diaflo XM-300 membrane. An anti-β-crystallin immunoadsorbent reacting with complexes from mature lens tissue yielded a non-filterable α-crystallin and a filterable γ-crystallin in the supernatant, indicating an absence of direct α-γ complexes. These results point to a potentially central role of β-crystallins in complex formation among lens proteins.     

Decreased association of aged alpha crystallins with gamma crystallins

Previous studies have demonstrated non-covalent interactions of alpha crystallins with gamma crystallins, under true equilibrium conditions. These interactions could affect short-range interactions of lens crystallins that are necessary for the transparent properties of the lens. Since the transparent properties of the lens decrease during aging, it is possible that there are corresponding changes in the ability of aged alpha crystallins to interact with gamma crystallins. In the following study, alpha crystallins were prepared from fetal and aged bovine lenses, then tested for binding to gamma crystallins using microequilibrium dialysis. The results demonstrate that during aging of the normal bovine lens, there is a decrease in the ability of alpha crystallins to bind to gamma crystallins, consistent with the involvement of this interaction in the transparent properties of the lens.     

Proteins associated with the membrane surface of lens fiber cells

The urea-soluble proteins (USP) from dogfish lenses were studied by high-resolution analytic methods. Immunoprecipitates were dissolved and analyzed in 8 m-urea-Tris-glycine buffer by polyacrylamide gel electrophoresis. All USP components were found to have properties in common with the water-soluble lens proteins (WSP) as characterized by Ferguson plots. Water-soluble lens proteins showed a preferential affinity for the isolated membrane ghosts of lens and red blood cells. Species differences are noted which may be related to species-specific functional requirements of the lens.     

Amino acid transport and crystallin synthesis in the bovine lens

Bovine lenses were incubated in a defined, bicarbonate-free culture medium (EMEM) and the kinetics of amino acid uptake and protein synthesis investigated. The kinetics were interpreted in terms of a simple multi-compartment model. [¹⁴C]tyrosine was found to be totally exchangeable in the incubated lens and the rate constant for the exponential increase in activity was 0·0175 hr^?1. The rate of influx was markedly reduced by incubating in the presence of ouabain (10^?5m), which also caused a concomitant disturbance of the normal sodium and potassium distributions.The soluble proteins from the ineubated lenses were fractionated on Sephadex G-200 and the rate of incorporation into the crystallins was shown to fall into two classes. The rate of synthesis of α and β_L crystallin was relatively rapid (rate constants approximately equal to 0·004 hr^?1), while the synthesis rates of β_H and $\text{β}{}_{\mathrm{<mtext>S</mtext>}}\text{γ}$ were both much slower (0·001 hr^?1).The efflux kinetics of [¹⁴C]tyrosine were determined and the rate of decrease of the free amino acid pool was identical to the rate of increase determined from an influx experiment. Hence the lenses are in a steady state with respect to free tyrosine throughout the incubation period (up to 160 hr). All classes of proteins continued to be synthesized during efflux experiments and there was no evidence for a breakdown of α or β_L crystallin during the time-course of these experiments.Ouabain slowed the rate of loss of tyrosine from the free amino acid pool, and this was interpreted in terms of an ouabain-induced decrease in synthesis rate rather than as a decrease in efflux rate from the lens. There was in fact a very marked decrease in the incorporation of [¹⁴C]tyrosine into the α and β_L crystallins on exposure to ouabain, and this decrease was apparent before and change in activity in the amino acid pool.     

Immunochemical studies on lens protein-protein complexes. II. On the mechanism of alpha-beta-gamma crystallin complex formation

An analysis of the β- and γ-crystallins present in various lens protein complexes of α-β, α-β-γ or only β-γ crystallins was done. This analysis was based on a detailed study of different proteins in the β- and γ-crystallin fractions.Beta and γ-crystallins were isolated from the different complexes by reacting each of them with anti-β crystallin antibodies used as ligands in an immunoadsorbent. The reaction resulted in dissociation of the complexes and binding of the various β-crystallins to the immunoadsorbent, leaving α- and γ-crystallins free in solution. After elution and concentration, the β-crystallins were compared in immunodiffusion tests with the high and low molecular weight β-crystallins. The freed γ-crystallins were first separated from α-crystallin by filtration on Diaflo XM-300 membranes. The filtrates were concentrated and compared with high and low molecular weight γ-crystallins.All of the β-crystallins which complexed with α-crystallin to form the α-β as well as the different α-β-γ crystallin complexes were found to contain β-crystallin molecules I and II, molecules which are typical of high molecular weight β-crystallin fractions. In addition, none of these β-crystallins contained β-crystallin molecule VII which is typically found in low molecular weight β-crystallin fractions. By contrast, all of the β-crystallins which did not complex with α-crystallin but which did form various β-γ crystallin complexes were related to the low molecular weight β-crystallins, as shown by the presence of β-crystallin molecule VII in these complexes and the absence of β-crystallins I and II. β-Crystallin molecules III through VI present in both high and low molecular weight β-crystallin fractions were found in all complexed crystallins. All complexes were also found to contain the same γ-crystallin molecules I and II.The data indicate that with progressing lens age, an initial α-β complex binds free γ-crystallins forming α-β-γ crystallin complexes. The latter complexes are not formed by association of α-β with β-γ complexes.     

Chaperone activity in the lens

Introduction : α‐crystallin, the major protein of the eye lens, is a molecular chaperone that is able to prevent the precipitation of denatured proteins. This activity is thought to be important for the maintenance of lens transparency. Loss of the activity has been postulated to contribute to the development of cataract. The purpose of this study was to determine how chaperone activity was affected by growth and ageing of the lens. Methods : α‐crystallins were purified from nine concentric tissue layers removed from an adult bovine lens. The ability to inhibit the precipitation of ß_L‐crystallin, following thermal denaturation, was used to assess the chaperone activity of these proteins. The molar ratio of α‐crystallin/ß_L‐crystallin required to inhibit the precipitation of ß_L‐crystallin by 50 per cent was used as a measure of the affinity of the chaperone for denatured protein. Results : As evidenced by a gradual increase in the ratio, from 0.52 to 1.24, the protective ability of α‐crystallin decreased from the outside of the lens into the centre, α‐crystallin from the cortex of the lens provided greater protection against precipitation of proteins than older α‐crystallin from the nucleus. The reasons for this were investigated. Gel electrophoresis of the proteins from each concentric layer revealed an increase in degraded polypeptides from approximately one per cent in the cortex to more than nine per cent in the centre of the lens. This increase appears to be correlated with the decrease in chaperone ability. Renaturing α‐crystallin obtained from the nucleus did not increase its chaperone activity, indicating conformational changes were not responsible for the decreased activity. Phosphorylation did not appear to have any significant effect on the chaperone activity. Conclusion : The loss of chaperone activity, accompanying fibre cell compression into the centre of the lens, can be attributed to degradation of the α‐crystallin polypeptides.