Hydration properties of lens crystallins

The water binding properties of bovine lens crystallins, alpha, beta H, beta L and low molecular weight (LMW), were investigated with different techniques. The water sorptive capacity was obtained in high vacuum sorption experiments volumetrically, and also gravimetrically in controlled atmosphere experiments. The two methods gave reproducible isotherms. The strongly sorbed water (bound water) was evaluated from the B.E.T. monolayer and from the values of isosteric heats. The bound water content of the crystallins were also measured by combined differential scanning calorimetry and thermogravimetric analysis. The non-freezable water contents calculated as % of the total water content and also as g g-1 crystallin, were used as an indicator for bound water. In all three different techniques, the following order was obtained: beta H greater than alpha greater than beta L = LMW.     

Ontogeny of human lens crystallins

The soluble proteins from prenatal and neonatal human lenses were fractionated by gel filtration into four distinct size classes viz. high molecular weight alpha-crystallin (HM-alpha), alpha-crystallin, intermediate molecular weight (IMW) proteins and low molecular weight (LMW) proteins. Extinction coefficients of the isolated proteins were determined and used to calculate the proportions of each fraction on a weight basis. The distributions of polypeptides within each of these fractions were analyzed by SDS gel electrophoresis and isoelectric focussing, followed by densitometric scanning of the gels. HM-alpha is detectable as early as the 14th week of gestation and its proportions increase rapidly, to about 9% of the total protein in the 1 year postnatal lens. The alpha-crystallin, IMW and LMW fractions show concomitant decreases and by 1 year they represent about 34, 35 and 18%, respectively. However, the proportions of IMW and LMW proteins do not accurately reflect those of the beta- and gamma-crystallins, as is often assumed. Substantial levels of non-crystallin polypeptides were found in the IMW protein fractions, including a group of very basic polypeptides (VBP) which comprised up to one-third of this material in the youngest lenses. Moreover, in postnatal lenses beta s-crystallin accounted for almost half of the LMW proteins. These points considered, alpha-crystallin is the major protein in the neonatal lens (approximately 42%, including HM-alpha), followed by the beta-crystallin (approximately 36% at most and probably less), the gamma-crystallins (approximately 11%) and beta s-crystallin (approximately 9%). Substantial changes in the proportions of specific polypeptides were observed throughout early development. These appear to result from changes at the level of protein synthesis and from postsynthetic modification. The A:B subunit ratio of alpha-crystallin drops from about 12 to below 3 during early development. This coincides with increasing levels of various deamidated and degraded subunits. The major beta-crystallin polypeptide also undergoes rapid deamidation and evidence is presented suggesting that the gamma-crystallins are subject to similar modification. The most dramatic changes were observed in the constituents of the LMW proteins. The synthesis of gamma-crystallins virtually ceases at some time around birth. At the same time, the levels of beta s-crystallin undergo an explosive increase. These and other changes are discussed in terms of their possible functional significance. They are also related to the complex protein status found in old lenses.     

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.     

Differential response of lens crystallins and corneal crystallins in degenerative corneas

Corneal degenerations, occurring either spontaneously or as a complication to other diseases, cause vision problems by endangering corneal transparency. Our past cornea research projects involving mice revealed that some recruited mice presented corneal problems similar to human corneal degeneration. The present study examines the histology of diseased mice corneas, including ultrastructure. Genome-wide microarray and proteomic methods were utilized to screen for molecular changes in the diseased corneas. It was found that abnormalities affected mainly anterior layers of the corneas. The most often observed histological abnormalities included neoplasm or detachment of the epithelial layer, erosion or breakage of Bowman membranes, blood vessel formation, and bleeding in the stroma. Microarray assay showed that among the 46 up-regulated probes in diseased corneas, 13 were for lens crystallins. However, all corneal crystallins genes remained unchanged. αA-crystallin was among the proteins that showed the greatest increase in diseased corneas, as detected by gel electrophoresis. We propose that lens crystallins, rather than corneal crystallins, are involved in the pathological process of corneal degeneration. Further study along these lines would provide insight into the mechanism of corneal transparency.     

Glycation-induced crosslinking of calf lens crystallins

To investigate the possible role of glycation in the onset of diabetic cataract we used calf lens crystallins as a model. After incubation with reducing sugars, the proteins were investigated by high-pressure gel permeation chromatography, SDS-PAGE and analytical ultracentrifugation. Glucose-6-phosphate incubation resulted in an increase in mean molecular weight of all crystallin fractions and the occurrence of high-molecular weight material, partly formed by disulphide bonds. The glycated crystallins showed a decrease of tryptophan fluorescence and an increase of a specific non-tryptophan fluorescence. This fluorescence was, however, not exclusively associated with the high molecular weight protein, but was present in all protein fractions.     

The precipitation and cross-linking of lens crystallins by ascorbic acid

Bovine lens beta-crystallin was incubated with increasing concentrations of sugars and sugar derivatives for a period of 2 weeks in the dark at 37 degrees C. Marked protein precipitation and a browning reaction was observed with both ascorbic acid (ASA) and dehydroascorbic acid (DHA), but little or no reaction was seen with several other sugars and sugar analogs. Similar incubations were carried out with 20 mM ASA, 20 mM DHA and 20 mM glucose, but with increasing amounts of the individual crystallins. Glucose was capable of precipitating gamma-crystallin in the presence of air, but this reaction was decreased if dithiothreitol and a chelating agent were added prior to incubation. ASA and DHA produced precipitation and browning with gamma- and beta-crystallin, but not with alpha-crystallin or lens soluble proteins. Similar reactivities were observed both in air and under reducing conditions. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of these reaction mixtures showed little or no cross-linking with any of the lens proteins by glucose. ASA and DHA caused detectable dimer formation with gamma-crystallin, but produced the formation of dimers as well as highly polymerized proteins at the top of the gel with all the other crystallins and with lens soluble proteins. A time-course experiment with alpha-crystallin in the presence of air showed no cross-linking with 100 mM glucose over a 6-week period; however, 10 mM ASA caused definite cross-linking at 2 weeks, and at 6 weeks a dark smear of protein was visible throughout the gel. ASA was still capable of inducing cross-linking under low oxygen conditions but the protein smearing was markedly diminished. Further, the cross-linking pattern was similar to that seen in the water-insoluble fraction from older human lenses and cataracts. This reaction may be significant in vivo because cross-linking was observed under low-oxygen conditions with as little as 2 mM ASA, which is the level of ASA normally present in human lenses.     

Interaction of lens crystallins with lipid vesicles

Previous studies have demonstrated that alpha-crystallin, but not beta or gamma-crystallin, can bind to lens membrane in a specific and saturable manner. To determine which components of the lens membrane might be involved in this interaction, each of these crystallins was incubated with reconstituted vesicles containing either phosphatidylethanolamine (PE), phosphatidylcholine (PC), or sphingomyelin (SPH). Alpha-crystallin, but not beta- or gamma-crystallin, bound to these vesicles in a saturable manner, in similar amounts as lens membrane. Together, these results suggest that the lipid moiety of the lens membrane may be involved in recognition of the alpha-crystallin molecule.     

Alkylation of rat lens crystallins with iodoacetamide

Alkylation of rat lens proteins with iodoacetamide during homogenization had no effect on protein disulfide content or on the proportions of soluble and insoluble lens protein. These results were similar for lenses, from young or older animals. With the exceptin of the β_H-crystallin fraction. alkylation yielded a normal crystallin population as determined by gel-permeation chromatography. Although each crystallin fraction reacted with iodoacetamide in proportion to its free sulfhydryl content, only the alkylated β_H-crystallin demonstrated an apparent reduction in molecular size so that it co-chromatographed with β_L-crystallin. Molecular weights of subunits for both β_H- and β_L-crystallin were modified by alkylation. Isoelectric focusing further revealed bands of higher isoelectric pH after alkylation. We conclude that subunit interactions which result in the protein assemblies, characterized as crystallins, do not depend on disulfide formation during isolation, and that β_H-crystallin has unique conformational or subunit properties which are sensitive to modification of the free-sulfhydryl group.     

Isoelectric focusing and immunochemistry of lens crystallins

This is a review of 11 published articles with some conclusive results on the isoelectric focusing and immunochemistry of lens crystallins. The techniques used throughout the work are isoelectric focusing and immunological methods.

The isoelectric focusing technique is performed on an analytical scale to realize the separation of proteins by isoelectric focusing in thin-layer polyacrylamide gels. The importance of carrying out all isofocusing techniques at 4 ° C, and also of measuring the pH gradients established at the same temperature is noticed. Free isoelectric focusing is used as a preparative step to isolate uncontaminated crystallin fractions. Both analytical and preparative isoelectric focusing experiments of crystallins performed throughout the work are in absence of urea or any other dissociating medium.

The purity of proteins isolated by free isoelectric focusing is examined by criteria of homogeneity which are founded on three different techniques, e.g. thin-layer isoelectric focusing, immunoelectrophoresis and antigen/antibody crossed electrophoresis.

The existence of various crystallins in the chick lens, like α-, β-crystallin and FISC, related to own data, is reviewed and nomenclatures of chick lens crystallins are compared with present data from literature. The antigenic properties of proteins from chick intra-ocular tissues are also discussed. Anti-total iris serum contains antibodies against crystallins and serum proteins. The presence of proteins immunologically identical to lens crystallins in chick iris is shown by indirect immunofluorescence experiments: brilliant fluorescence of the lens is observed due to the presence of crystallin antibodies in anti-total iris serum. It is concluded that proteins of all intra-ocular tissues which are immunologically identical to lens crystallins are essential structural elements. Contrary to this statement several authors support the theory that the presence of proteins immunologically identical to lens crystallins in the intra-ocular tissues is due to a post-mortem diffusion of these antigens from the lens. The presence of several erum proteins in all intra-ocular tissues excepting lens is shown. Evidence is presented that the number of serum proteins in intra-ocular tissues is restricted by the various blood/ocular barriers, and that the blood/retinal barrier is of the same nature as the blood/brain barrier.

The presence α-, β-, γ- and pre-α-crystallins is shown in various stages of the bovine embryonic lens by thin-layer isoelectric focusing and various immunological methods. It is defined that the bovine γ-crystallins have isoelectric points higher than pI = 7.0. It is shown by immuno-osmophoresis that the embryonic α-crystallins consist of two molecular forms with different electrophoretic mobilities. The β-crystallins are forming half the total amount of the crystallins present in the embryonic lens. It is concluded that the γ-crystallins are more prominent in embryonic cow lens and in adult lens nucleus and less prominent in adult lens cortex. The γ-crystallin composition of embryonic bovine lenses and of adult lens nucleus appears to be different from those of the adult lens cortex due to a low concentration of some γ-crystallin components in the lens cortex. Moreover, the concentration of γ-crystallin in embryonic lens extract and in the lens nucleus is around 20 %, and in the lens cortex extract about 1%, as determined by thin-layer isoelectric focusing and by free isoelectric focusing.

A comparison is made of changes in the bovine embryonic lens during development : a slight increase of α-crystallin, a gradual increase of β-crystallins and a slight decrease of γ-crystallins is observed.

As determined by quantitative immunodiffusion experiments by Rabaey and collaborators, the embryonic γ-crystallin in the lens reaches a maximum concentration at an embryonic stage of 2.6 month, followed by a rapid decrease before birth. The concentration of the embryonic γ-crystallin in the adult lens goes down with aging to a constant level. This altogether stamps the embryonic γ-crystallin to an embryonic protein by pre-eminence.

The composition of crystallins from larval amphibian stages and adult Xenopus laevis is studied by thin-layer polyacrylamide isoelectric focusing. The lens of the larval stages contains a high amount of γ-crystallins which decreases gradually. The β-crystallins show a gradual increase during embryonic development, while the α-crystallin is very low in concentration in the larval stages. The adult X. laevis lens shows a decrease of certain γ-crystallin components, an increase of α-crystallin concentration, while the β-crystallins are the major fraction.

A comparison is made of the protein composition of chick and pigeon total lens and of the bovine embryonic and adult cortical and nuclear lens, with special reference to the γ-crystallins, by thin-layer isoelectric focusing. It appears that the concentration of crystallins with isoelectric points over 7 in chick and pigeon total lens and in cow lens cortex is very low, contrary to those from the bovine embryonic and nuclear lens. The FISC protein is prominent in the chick lens, lower in concentration in the pigeon lens and absent in bovine lens. The α-crystallin concentration in adult chick lens is lower than in the cow lens cortex. The β-crystallins in all lenses form the major proportion of about half to three-quarter of the crystallins present in all species compared.

     

Isoelectric focusing of embryonic cow lens crystallins

Soluble lens crystallins from several embryonic stages and also from adult cow lens cortex are compared by thin layer isoelectric focusing, immunoelectrophoresis, immuno-osmophoresis and antigen/antibody crossed electrophoresis. A gradual change in crystallin composition is found between the lens proteins of 1·5–7 month embryos.Thin layer isoelectric focusing studies of various embryonic stages from 1·5 to 7 months show a decrease of γ-crystallins along with an increase of β-crystallins and a slight increase of α-crystallins. When compared with the adult lens nucleus, the cortex shows a sharp decrease in concentration of γ-crystallin components. In addition, the composition of γ-crystallin of embryonic lens is similar to that of the adult lens nucleus, but different from that of the adult lens cortex. This difference appears due to certain components of γ-crystallin that are absent or present in lower amounts in the adult lens cortex compared with the embryonic lens. The same number of α-, β- and γ-crystallin antigens are present, they differ only in the relative concentration of the protein components.In all embryonic stages two pre-α-crystallins could be detected. These pre-α-crystallins have a high anodic mobility and appear to have a molecular weight lower than α-crystallin itself; the pre-α-crystallin is found to be eluted with the low molecular weight β-crystallins in Sepharose gel chromatography.By gel chromatography procedure, crystallins of cow lens cortex could be separated into five fractions; α₁-crystallins of a molecular weight > 2,000,000, α₂-crystallins, two β-crystallin fractions and γ-crystallin. Immuno-osmophoresis analysis shows that α₁-crystallin is present in higher amounts in embryonic lens, and in adult lens nucleus it is higher than in adult lens cortex, while the α₂-crystallin is present both in cortex and nucleus.     

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.     

Purification and some properties of camel lens crystallins

Five major crystallin fractions were found in camel lens fractionated by Sepharose CL-6B column chromatography. These crystallin fractions were named alpha high (alpha H), alpha low (alpha L), beta high (beta H), beta low (beta L) and gamma (gamma) by comparison with the elution profiles and molecular weights of rabbit crystallins. The amino acid composition and isoelectric focusing bands for crystallins of camel and rabbit were remarkably similar, but individual differences were found. By means of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), two alpha H crystallin subunits, of 23K and 20K molecular weight, were found in camel and rabbit. Likewise, a single 21K molecular weight band was found in the gamma crystallin of camel and rabbit. In camel, the beta high crystallin consisted of five major subunits, while rabbit beta high crystallin consisted of only three subunits. On SDS-PAGE, camel and rabbit beta low crystallins both showed two major subunits of 27K and 23K molecular weight but camel beta low crystallin showed an additional 35K molecular weight subunit. Characterization of camel lens crystallins may contribute to understanding the effect of aging on aggregation of camel crystallins.     

Bendazac decreasesin vitro glycation of human lens crystallins

Bendazac has been used as an anti-cataractogenic drug. It has been reported that this acts by preventing protein denaturation. In this study the ability of bendazac to inhibitin vitro glycation of human lens crystallins was evaluated. Possible effects of bendazac were detected by incubation of WS crystallins with the reducing sugars glucose and fructose. The efficiency of bendazac was evaluated by means of selected parameters including: browning, glycation (measured as tyrosine content) and specific NTP-fluorescence. The results showed clearly that bendazac (bendazac L-lysine and sodium) inhibits the early stages of protein glycation, as well as the formation of fluorescent advanced glycation products. Bendazac lysine (20 mM) proved to be more effective in inhibiting fluorescence development (67% inhibition) than the corresponding sodium salt (35% inhibition). No significant differences were found with respect to furosine levels; about 40% inhibition was produced with either bendazac lysine or sodium salt bendazac clearly inhibits glycation of human lens crystallins, as can be efficiently monitored by following specific changes in lens protein fluorescence. These results may constitute a new and relevant therapeutic approach to monitoring cataract development.     

Binding of actin to lens alpha crystallins.

Actin has been coupled to a cyanogen bromide-activated Sepharose 4B column, then tested for binding to alpha, beta, and gamma crystallin preparations from the bovine lens. Alpha, but not beta or gamma, crystallins bound to the actin affinity column in a time dependent and saturable manner. Subfractionation of the alpha crystallin preparation into the alpha-A and alpha-B species, followed by incubation with the affinity column, demonstrated that both species bound approximately the same. Together, these studies demonstrate a specific and saturable binding of lens alpha-A and alpha-B with actin.     

Spectroscopic studies on the interaction of calf lens membranes with crystallins.

The interaction of crystallins with lens membranes and liposomes was studied by fluorescence and circular dichroism (CD) measurements. Two extrinsic fluorescence probes ANS (1-anilino-naphthalene-8-sulfonic acid) and DPH (1,6-diphenyl, 1,3,5-hexatriene) were used to detect the binding and to explore the binding site. The ANS fluorescence intensity is greater in membranes than in liposomes, but is reverse for DPH. Among alpha, beta and gamma-crystallins, only alpha c-crystallin decreased the ANS fluorescence intensity in membranes, indicating a binding between membranes and alpha c-crystallin. The binding site appears to be at the polar-apolar interface in membrane protein (MIP26) and alpha c-crystallin. Fluorescence polarization measurements show that lipid bilayer becomes less mobile with alpha c-crystallin binding. The change in the near UV CD due to the binding also indicates a decreased freedom of rotation of aromatic amino acid residues either in MIP26 or in alpha-crystallin.     

Differential effects of galactose-induced cataractogenesis on the soluble crystallins of rat lens

Soluble lens crystallins from 6-10-week-old, galactose-fed, male Sprague-Dawley rats were analyzed by two-dimensional polyacrylamide gel electrophoresis at each of the five Sippel stages of cataractogenesis. Electrophoretograms were compared with similarly analyzed crystallins from comparably aged, chow-fed controls. Polypeptides were assigned to crystallin families and subfamilies on the basis of chromatographic fractionations with Sephadex G-200, superfine. Staining intensities of polypeptides from control lenses remained essentially unchanged throughout the experimental period, while those of the polypeptides from cataractous lenses showed non-uniform changes. Staining of the genomic gamma-crystallins increases up to at least stage 3; by stage 4, staining of gamma-chains, with perhaps those of gamma 5 and gamma 6 excepted, diminishes and in the total cataract, staining of all chains is further reduced. With possibly the addition of one chain, the total number of postsynthetically modified gamma-crystallins in cataractous lenses does not exceed that in the comparably aged normal lens. The genomic alpha- and beta-crystallin polypeptides are sustained close to normal levels up to stages 3 or 2, respectively, after which their gradually falling levels are accompanied by the generation of new species or elevated levels of existing post-translational species. An exception to this behavior is the rapid and total loss of beta B1a, a genomic subunit implicated in the aggregation of beta H-crystallins. Charge heterogeneity and variable pI displayed by beta B1a and other highly cationic beta- and gamma-crystallin polypeptides can be induced during isoelectric focusing and may be due to thiol group oxidation.     

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.     

Differential synthesis of crystallins in the developing rat eye lens

The patterns of protein synthesis in rat lenses ranging in age from newborn to 4 months were compared. After incubation of lenses in [35S]methionine-containing medium it was possible to identify the de novo synthesized crystallins by two-dimensional gel electrophoresis and fluorography, in combination with peptide mapping and immunoblotting. It was found that the relative synthesis of alpha A and beta A3 stays fairly constant in rat lenses of all investigated ages. The relative synthesis of beta B2 and gamma s shows a pronounced increase with age in these post-natal lenses. A differential decrease can be observed in the relative synthesis of the other six gamma-crystallins (gamma A-gamma F). There appears to be a good correlation between the changes in relative synthesis of the various crystallins and previously reported alterations in mRNA levels, although certain mRNAs exhibit marked differences in translational efficiency.