Hydration properties of the molecular chaperone alpha-crystallin in the bovine lens.

Topographic studies of crystalline fractions from different morphological layers of the young adult bovine lens were conducted. Crystallin profiles were obtained for each lens layer, using thin-layer isoelectric focusing in polyacrylamide gel (IEF). Water soluble (WS) crystallins from the lens equator revealed a separation into HM (high molecular weight) alpha(L)-, beta(H)-, beta(L)-, beta(S)-, and gamma-crystallins. The nature of the water insoluble (WI) protein fraction in the separated lens layers reflected the aggregated state of alpha(L)-, beta(L)-, beta(S)-, and gamma-crystallins in different regions of the lens, concealed in the central cavity of the alpha-crystallin chaperone model. The IEF data demonstrate a possible chaperone-like function for alpha-crystallin in the nucleus and inner cortex of the lens, but not in the outer cortex. The water binding properties of bovine lens alpha-crystallin, calf skin collagen, and bovine serum albumin (BSA) were investigated with various techniques. The water adsorptive capacity was obtained in high vacuum desorption experiments volumetrically, and also gravimetrically in controlled atmosphere experiments. The NMR spin-echo technique was used to study the hydration of protein samples and to determine the spin-spin relaxation times (T(2)) from the protons of water adsorbed on the proteins. Isolated bovine lenses were sectioned into 11-12 morphological layers (from anterior cortex through nucleus to posterior cortex). The water content in relation to dry weight of proteins was measured in individual morphological lens layers. During water vapor uptake at relative humidity P/P(0) = 0.75, alpha-crystallin did not adsorb water suggesting that hydrophobic regions of the protein are exposed to the aqueous solvent. At relative humidity P/P(0) = 1.0, the adsorption of water by alpha-crystallin was 17% with a single component decay character of spin echo (T(2) = 3 msec). Addition of water to alpha-crystallin to about 50% of its weight/weight in the protein sample showed T(2) = 8 msec with only one single component decay of the spin-echo signal. The single component decay character of the spin echo indicates water tightly bound by alpha-crystallin. Under a relative humidity P/P(0) = 1.0, collagen and BSA adsorbed, correspondingly, 19.3 and 28% of water and showed a two-component decay curve with T(2) about 5 and 40 msec. The findings demonstrate the presence of two water fractions in collagen and BSA which are separated in space. The IEF data suggest a tight binding of water with alpha-crystallin with similar distribution patterns in the lens layers. To conclude, it was found that alpha-crystallin can immobilize water to a greater extent than other proteins such as collagen and BSA. These results shed new light on structural properties of alpha-crystallin and its superhydration properties and have important implications for understanding the mechanism of the chaperone-like action of this protein in the lens and non-ocular tissues.

Analysis of tear proteins by one- and two-dimensional thin-layer iosoelectric focusing, sodium dodecyl sulfate electrophoresis and lectin blotting. Detection of a new component: cystatin C.

BACKGROUND: Isoelectric focusing (IEF) of tear proteins has not yet been carried out in a satisfactory way. Two-dimensional (2D) electrophoresis, especially in the combination of IEF with SDS, is able to differentiate between proteins in detail. The purpose of this study was therefore to analyze tear proteins by 1D IEF alone and in combination with a 2D pattern, and by IEF followed by lectin staining. METHODS: Ampholines, covering a broad range from pH 3 to pH 10, were applied. After IEF, semi-dry blotting and incubation with a group II lectin and two group V lectins was performed. RESULTS: Tear proteins could be separated into 31 single bands. Tear-specific pre-albumin (TSPA), lactoferrin, sIgA, IgG and lysozyme were found to be main components. Isoelectric points (IEPs, pls) of all proteins separated were determined by comparison with IEF standards. 2D patterns of IEF and SDS electrophoresis were obtained for the main subunit components of lactoferrin, sIgA, TSPA, and lysozyme. An additional new component of considerable concentration was focused at pI 8.6 with a subunit MW of 14 kDa. With s-WGA a component at an IEP of 5.2 was visualized, representing transferrin. With SNA, lactoferrin stained as a sharp main band at pI 5.1 with three additional weaker bands at IEPs from 4.8 to 4.9. At IEPs between 4.4 and 6.1, multiple components of sIgA were stained with MAA. The sugar specificity of transferrin at pI 5.2 was beta-GlcNAc. Lactoferrin showed glycation with NANA-alpha-2-6-Gal or NANA-alpha-2-6-GalNAc, whereas the sugar specificity of sIgA was NANA-alpha-2-3-Gal. CONCLUSIONS: The investigative strategy applied here, including IEF alone, in combination with SDS-electrophoresis, and SDS-electrophoresis followed by lectin staining proved to be a reproducible method for tear protein analysis of hitherto unexperienced capacity. Lectin-stained bands of native tear proteins are not uniformly glycated by one sugar residue, but show various sugar specificities. IgA as a whole molecule is specifically glycated with NANA-alpha-2-3-Gal.

Higher glycation of beta L- and beta S-crystallins in the anterior lens cortex and maximum glycation of gamma-crystallins in the bovine lens nucleus, demonstrated by frozen sectioning, isoelectric focusing and lectin staining.

The aim of the current study was to demonstrate glycation of beta L-, beta S- and gamma-crystallins in the young bovine lens. To establish which of the crystallins are glycated and where they are located in the lens, we carried out microsectioning of the lens, followed by isoelectric focusing (IEF). Four bovine lenses of 1.183 +/- 0.070 years were frozen-sectioned into equator and 11 layers. Water-soluble crystallins were separated by IEF and stained: (1) with Coomassie brilliant blue for proteins; (2) with the lectin concanavalin A, followed by horseradish peroxidase and diaminobenzidine, for glycated proteins. Experiments were performed with crystallins and proteins in native form, in the absence of denaturants. The crystallins were separated by IEF into alpha-crystallins of high molecular weight (HM), alpha L-, beta H-, beta L-, beta S- and gamma-crystallins. In the lectin staining experiments, only HM, beta L-, beta S- and gamma-crystallins were positive, whereas the alpha L- and beta H-crystallins were negative. Contrary to the glycated gamma-crystallins in the lens nucleus, the beta S- and beta L-crystallins were predominantly glycated in the anterior cortex and to a somewhat lower extent also in the posterior cortical regions. The degree of glycation (total densitometric readings of lectin-stained bands/Coomassie-blue-stained bands) is as follows: total gamma-crystallins 2.44, beta S-crystallins 0.77 and beta L-crystallins 0.28. Though glycation in the bovine lens is very low, lectin staining is sufficiently sensitive to detect the various glycated crystallins. The degree of glycation of gamma-crystallins was 3 times higher than that of beta S-crystallins and 9 times higher than that of beta L-crystallins.

Detection of glycated fetal human lens crystallins by concanavalin-A and aldehyde staining.

The binding of the lectin Concanavalin-A (Con-A) to crystallins was investigated. For this purpose, human fetal and juvenile lens crystallins were isofocused and specifically stained brown for glycoproteins by the lectin Con-A, and purple by the periodic acid Schiff's reagent (PAS). In reference experiments protein bands were stained with Coomassie Blue for proteins. Since Con-A is a protein with glucose-specific receptors, the following crystallins, glycated with this sugar, were visualized after isoelectric focusing: HM-, beta L-, beta S- and gamma-crystallins, but not alpha L- and beta H-crystallins. Glycation increased significantly with fetal age. The crystallins themselves could also function as sugar receptors, because it was shown that they possessed also receptors for glucose, like Con-A. This crystallin receptor staining revealed beta L-, beta S-, gamma- but hardly HM-crystallins. The PAS, Con-A and receptor stainings gave in principle identical results. The glycoproteins Con-A, horseradish peroxidase and lentil lectins were used as positive controls.

The glycation of bovine lens betaL-, betaS- and gamma-crystallins demonstrated by isoelectric focusing and lectin staining.

The aim of the current study is to detect glycation of betaL-, betaS- and gamma-crystallins in the young bovine lens. To determine which of the crystallins are glycated, we have made isoelectric focusing of the water-soluble crystallins of four bovine lenses of 1. 183+/-0.070 years. Samples are stained: (1) with Coomassie Brilliant Blue for proteins; (2) with the lectin Concanavalin-A, followed by horse-radish peroxidase (HRP) and diaminobenzidine (DAB). Experiments are performed with crystallins in native form, in absence of denaturants. The crystallins are separated by isoelectric focusing into: alpha-crystallins of high-molecular weight (HM)-, alphaL-, betaH-, betaL-, betaS- and gamma-crystallins. In the lectin staining experiments only HM-, beta L-, betaS- and gamma-crystallins are positive, whereas the alphaL- and betaH-crystallins do not stain. Though glycation in the bovine lens is very low, lectin staining is sufficiently sensitive to detect the various glycated crystallins.

Comparative investigations on water-soluble crystallins of the embryonic, fetal, and postnatal human lens during development and ageing.

To compare the crystallin composition of embryonic and fetal human lenses with those of postnatal and adult lenses, we investigated the crystallins of lenses of various ages (from the 5th gestational week to 55 years) by gel chromatography, isoelectric focusing, immunodiffusion, and immunoelectrophoresis. Age-related changes were calculated as relative percentages of the different classes and subclasses of crystallins. During prenatal lens development the percentages of both high- and low-molecular-weight alpha-crystallins as well as gamma-crystallins gradually increased, whereas the percentage of beta-crystallins decreased. A considerable change in crystallin composition was found immediately after birth: the relative percentage of beta-crystallins increased, whereas that of gamma-crystallins decreased. Gel-filtration analysis of crystallins from juvenile and adult lenses showed a high-molecular-weight peak, which was not found in extracts from fetal and new-born lenses.

Calf lens alpha-crystallin, a molecular chaperone, builds stable complexes with beta s- and gamma-crystallins.

Calf water-soluble (WS) crystallins consist of high-molecular weight (HM), alpha-, beta H-, beta L-, beta s- and gamma-crystallins. alpha-Crystallin as a molecular chaperone forms a structure with a central hole, known as Carver's model. The only crystallins fitting into this central cavity are beta s- and gamma-crystallins, with native molecular weights of 28 and 18.5-21 kD, respectively. The beta s-crystallin is loosely bound to this structure, whereas with the application of 7M urea in the sample, beta s-, may be some beta L-components and all gamma-crystallins emerge from the central hole. Although WS and water-insoluble (WI) crystallins display the same immunologic determinants, there is an appreciable difference in electrophoretic mobility already in the young calf lens. alpha-Crystallins from the WI part demonstrate a clear cathodic shift. WI beta- and gamma-crystallins show smaller but well perceptible cathodic shifts. The chaperone function of alpha-crystallin preserves the original structures of beta s- and gamma-crystallins for aggregation and other influences.

Isoelectric focusing of crystallins in microsections of calf and adult bovine lens. Identification of water-insoluble crystallins complexing under nondenaturing conditions: demonstration of chaperone activity of alpha-crystallin.

Topographic studies of crystallin fractions from the young adult bovine lens revealed that lenses do not have a homogeneous distribution of crystallins. There are, however, gradual differences between the cortices and the nucleus. The isolated lenses were separated mechanically into lens equator and inner cylinder. The latter was then sectioned in a special sectioning machine into 11-12 morphological layers (from anterior cortex through nucleus to posterior cortex). Matters of the lens sections were separated into water-soluble (WS) and water-insoluble (WI) crystallins. The WI fractions were solubilized with 100% formamide, or dissolved into 7 M urea. Crystallin profiles were obtained for each lens layer, using thin-layer isoelectric focusing in polyacrylamide gel. WS crystallins from the lens equator revealed a separation into HM-, alpha L-, beta H-, beta L-, beta S- and gamma-crystallins. The WI fractions of the layers dissolved in urea gave a separation into the individual HM- (3 components), alpha L- (4 components), beta- (6 component groups), beta S- (2 components) and gamma- (11 components) crystallins in the different morphological layers. The results confirm that a significant age-related increase in several beta- and gamma-crystallins incorporated into alpha-crystallins exists in the patterns of WI fractions of the different layers from lenses of 2.2 and 5.9 years. The WI crystallins solubilized in formamide showed only the presence of HM weight and alpha-crystallin moieties, due to the action of chaperone activity of alpha-crystallin. The nature of the WI protein fraction in the separated lens layers reflected to the aggregated state of: alpha L-, beta L-, beta S- and gamma-crystallins in the different regions of the lens, concealed in the central cavity of the alpha-crystallin chaperone model.

Basic biochemical parameters of one hundred cataractous lenses from Egyptian patients.

Cataractous lenses from an extended population of 100 Egyptian patients whose age, sex and case history had been recorded, were collected and classified morphologically. After determination of the lens wet weights, lenses were microsectioned and basic parameters such as wet weight, dry weight, water content and contents of water-soluble and water-insoluble crystallins of the samples were determined. The data obtained document moderate as well as drastic changes of the investigated parameters along with aging and cataractogenesis, and extend our knowledge from small numbers of hitherto investigated human lenses to a considerably high number of cases.

Distribution of water-soluble crystallins in microsectioned cataractous lenses from one hundred Egyptian patients.

Morphologically classified lenses from 100 cataract patients from the Mansoura Eye Hospital (Egypt) were microdissected and the crystallin composition and distribution were analyzed by thin-layer isoelectric focusing (IEF). The IEF profiles of cataractous lenses were compared with each other, with those of sclerotic lenses and with a normal lens profile. The alterations in the composition of high-molecular-weight (HMW)-, alpha-, beta H-, beta L-, beta s-, and gamma-crystallins along with normal aging, are superimposed by pronounced cataract-related changes which are different for the various types of cataracts. The general feature includes a continuous loss of gamma-, beta s- and beta L-crystallins of higher IEPs and an increase of HMW material. This is highly pronounced in the nucleus of nuclear cataractous lenses. In cortical cataractous lenses, changes start in the cortical layers. No differences could be observed between alterations in Caucasian lenses and this extended Egyptian lens population.

Staining of free sulfhydryl groups of proteins after separation by isoelectric focusing of Bio-Rad standards and lens crystallins.

In this study, we propose a new staining method for free sulfhydryl groups of proteins after having separated native samples by thin-layer isoelectric focusing (IEF) in absence of detergents. A comparison was made between proteins stained purple for free sulfhydryl groups (SH) and proteins stained blue by Coomassie blue (CB). A stainability factor, F = %SH/%CB, was calculated for each protein. The Bio-Rad IEF standards containing seven marker proteins for pH scale determination were stained purple, in the same way as they were designed for CB staining. To prove the validity of the currently proposed staining method for a defined protein system such as the eye lens crystallins, these proteins were also stained after IEF as described above. The factor F was calculated for all alpha-, beta-, and gamma-crystallin components that stained in both methods. We discovered that alpha-crystallins contained comparatively high amounts of free SH groups, while some beta- and gamma-crystallin components also contained considerable amounts of free SH groups. The SH staining procedure with 2,2'-dihydroxy-6,6'-dinaphthyl disulfide applied after IEF appeared to be useful, specific, and reproducible.

Molecular mass distribution of water-soluble crystallins from the human foetal lens during development.

The water-soluble crystallins of twenty human foetal lenses with gestational ages of 112-231 days were analysed by size-exclusion chromatography. The crystallin distribution showed similar patterns for all foetal lenses, but clear changes in the proportions of different crystallins were evident. The distribution showed that the water-soluble part of all the lenses already contained high-molecular-mass material. Also beta-crystallins of high molecular mass (beta H), formed by post-translational changes, were detected in all stages. During gestation, the percentage of high-molecular-mass crystallins and of alpha-crystallins of low molecular mass (alpha L) decreased significantly. The total beta-crystallins (beta T) and the total gamma-crystallins (gamma T) increased significantly. The low Mr crystallins were resolved into three peaks, designated beta s-, gamma H- and gamma L-crystallins. They increased significantly during development. These significant increases of the low Mr crystallins took place exclusively in the developing lens. The rate of protein synthesis of the low Mr crystallins was 23% of the total water-soluble crystallin synthesis rate.

Protein profiles of microsections of the fetal and adult human lens during development and ageing.

The water-soluble proteins of the human fetal lens (175- and 285-day-old) contain HM-, pre-alpha-, alpha-, beta- and gamma-crystallins. Using the frozen-sectioning technique, it can be demonstrated that the fetal lens does not have an homogeneous distribution of crystallins, but there are gradual differences between the cortices and the nucleus. The frozen-sectioning technique shows for the adult lens significantly increasing amounts of beta-crystallins of pI 4.95-5.55, especially at the posterior supra-nuclear layer, increasing amounts of HM-crystallins and decreasing amounts of beta-crystallins of pI 5.80-7.05 in the nucleus. This microsectioning technique was correlated with Scheimpflug photographs of the fetal and adult lens. In the fetal lens, the anterior capsule and 2 peaks in the anterior and posterior supranuclear layers could be visualized after densitometry. In the adult lens 5 layers could be demonstrated, e.g. the anterior capsule, the anterior supranuclear layer, the nucleus, the posterior supranuclear layer and the anterior capsule.

The protein distribution of bovine, human and rabbit aqueous humour and the difference in composition before and after disruption of the blood/aqueous humour barrier.

The protein distribution of bovine, monkey, dog, human and rabbit aqueous humour (AH) was determined by capillary isotachophoresis (ITF). The main component was albumin, and components of lower concentrations were transferrin, IgA and IgG. The results of the analysis by ITP were confirmed by immunoelectrophoresis. The appearance of the ITP patterns of normal AH's from 5 species was almost identical, the same main components were present in the AH of each species. When the blood/AH barrier was disrupted, the protein composition of the AH was changed abruptly. ITP was the technique of choice to determine quantitative changes in protein composition of the AH's. In rabbit secondary AH, ITP showed high proportions of albumin and enhanced concentrations of transferrin. Examples were given of the difference between early and late aspiration in bovine and rabbit. The ITP was thus the method of choice to confirm whether the blood/aqueous humour barrier was intact or damaged by the influence of a drug, given to experimental animals.

Crystallin profiles of calf and bovine lens microsections, stained for free sulfhydryl groups and proteins.

Calf and bovine lenses of 0.98 and 8.40 years old were separated mechanically into lens equator and inner cylinder. The inner cylinder was cut into 10 to 11 sections by a microsectioning device. These sections were investigated on the protein profiles of water-soluble crystallins, stained for proteins by Coomassie Blue (CB). These crystallins were also specifically stained purple for free sulfhydryl groups (SH). It appeared that all crystallins that were stained blue for proteins were also stained purple for sulfhydryl groups. This means that all crystallins contain free sulfhydryl groups. Going from anterior and posterior cortex to the nucleus of the lens, there was an appreciable increase of the percent of gamma-crystallins, whereas especially in the older lenses a decrease of gamma-crystallins could be observed in the lens equator and the anterior and posterior cortices. A stainability factor F = %SH/%CB was calculated for all crystallins. HM-, alpha- and beta s-crystallins exhibit high values of factor F. For the bovine lens, factor F of HM-crystallin displayed a maximum in the nucleus, whereas this factor decreased for gamma-crystallins towards the nucleus. This microsectioning technique allows for determining age-related differences between the sections obtained. This may lead to a comprehensive understanding of age-related changes within one lens, including cataractous changes.

Water-insoluble high-molecular-weight and alpha-crystallins as the source of the Scheimpflug light scattering pattern in the rat lens.

Lenses of 14-week-old rats were separated into 10 layers or fractions by a frozen-sectioning technique. The biochemical characteristics of these layers were assigned to corresponding parts of the densitometric reading obtained from the Scheimpflug negative, which enables a correlation of light scattering values recorded in vivo to protein patterns in the same area. Calculated as percentage of lens dry weight, all water-soluble crystallins show minima and the water-insoluble crystallins show maxima in the lens nucleus. This demonstrates that the nucleus contains the bulk of the water-insoluble high-molecular-weight and alpha-crystallins, being the source for the Scheimpflug light scattering pattern.

The quantification of eight enzymes from the ageing rat lens, with respect to sex differences and special reference to aldolase.

Eight enzymes, e.g. lactate dehydrogenase, malate dehydrogenase, fructose-diphosphate aldolase, sorbitol dehydrogenase, glucose-6-phosphate dehydrogenase, hexokinase, phosphofructokinase and pyruvate kinase were estimated quantitatively in the rat lens from 37 to 1,211 days of age, by spectrophotometric methods. The activity was expressed as mU/g LWW. All enzymes measured showed declining activities, but LDH, ALD, SDH, G-6-PDH, HK and PFK gave a significant decrease during ageing when plotted semi-logarithmically from 37 to 1,211 days. SDH and G-6-PDH showed a statistically significant difference between the enzymes from the male and the female lenses. The female lens always had a lower activity than the male lens. Of all enzymes the specific activity, expressed as mU/l mg protein, was calculated. This specific activity appeared to be rather constant during ageing, except for ALD. In the female lenses, the specific activity of 7 enzymes was lower than in the male lenses. For ALD the specific activity decreased significantly in the male lens from 5.32 at 37 days to 0.88 at 1,211 days. In the female lens this significant decrease was from 4.97 to 0.81.

Alterations of lens metabolism with experimentally induced cataract in rats.

Rat lenses with experimentally induced cataract (either by naphthalene or by streptozotocin) were analyzed biochemically. Both noxae had some effects in common. Water-soluble protein and aldose reductase activity decreased, and glucose-6-phosphate dehydrogenase, phosphofructokinase and glutathione reductase activity increased. A specific effect of streptozotocin was the rise in glucose, fructose and sorbitol. A specific effect of naphthalene was increased amounts of water-insoluble protein.