Structure/function studies of dogfish alpha-crystallin, comparison with bovine alpha-crystallin.

PURPOSE: alpha-Crystallin is the major protein of the mammalian lens where it contributes to the refractive properties needed for vision and possibly to the stability of the tissue. The aim of this study was to determine whether the properties of alpha-crystallin have changed during the course of evolution. METHODS: Dogfish alpha-crystallin, which appeared over 420 million years ago, has been contrasted with bovine alpha-crystallin, which emerged around 160 million years later, by comparing their sizes, the microenvironments of their cysteine and tryptophan residues, their chaperone-like activities and the flexibility of their COOH-terminal extensions. RESULTS: Dogfish alpha-crystallin consists of alphaA- and alphaB-polypeptides, in a 1:5 ratio, and has a molecular mass of around 400 kDa. By contrast, the bovine protein is around 600-800 kDa in mass and has a 3:1 subunit ratio. Cysteine residues in the proteins were equally accessible to reaction with 5,5'-dithiobis-(2-nitrobenzoic acid). Quenching of fluorescence with acrylamide indicated tryptophan residues in the two proteins were in similar environments. The chaperone activity of dogfish alpha-crystallin was comparable to that of bovine alpha-crystallin in preventing the heat-induced precipitation of beta(L)-crystallin but the dogfish protein was three times more effective at preventing insulin precipitation after reduction at 37 C. (1)H nuclear magnetic resonance spectroscopic studies showed that the last 17 amino acids of the dogfish alphaB polypeptide (V162-K178) have great conformational flexibility, are highly exposed to solvent and adopt little ordered conformation. This is comparable to, but slightly longer in length, than the COOH-terminal extension observed in mammalian alpha-crystallins. CONCLUSIONS: The structure and properties of alpha-crystallin have changed relatively little during the evolutionary period from the emergence of sharks and mammals.

Growth related changes to functional parameters in the bovine lens.

Dimensions, volumes and protein contents were measured for bovine lenses with wet weights ranging from 0.17-3.07 g (2 months gestation to 19 years post-natal). All increase in a non-linear fashion. The lens becomes flatter with age due to a more rapid increase in the equatorial plane, but the ratio of anterior to posterior sagittal distances remains constant (1.19). The radius of curvature increases from 4.9 to 15 for the anterior surface and from 4.4 to 13 for the posterior. Protein content increases more rapidly than volume resulting in an increased average protein concentration from around 18% in the early prenatal lens to nearly 50% in the 19 year old. Total protein content (TPC) was found to be related to wet weight (We) according to the equation, TPC = 0.3We1.33. It is suggested that TPC is a better parameter for describing growth than wet weight or age. The refractive index, in the equatorial plane, increases towards the centre, from 1.38 at the edge of the lens. The maximum index, in the centre, increases with lens size up to 1.474 in the largest lens studied. This corresponds to a protein concentration of 70%. In all lenses, refractive index and protein concentration gradients were superimposable when plotted from the outside towards the centre. The optical performance of the lenses was assessed by measuring the back focal length which increases gradually from 24 to 51.5 mm over the 0.17 to 3.07 g size range. This was attributed to the increased radii of curvature.

Oxidative changes in human lens proteins during senile nuclear cataract formation.

1. Proteins from the cortex and nucleus of the human lens were studied to determine if any changes could be detected in their amino acids during senile cataract formation. 2. Senile nuclear cataract formation was found to be accompanied by a progressive oxidation of cysteine and methionine. The oxidation of methionine and changes in the distribution of the nuclear proteins did not appear to start until about 60% of the cysteine had been oxidized. 3. In the advanced nuclear cataractous lens, about 90% of the cysteine has been oxidized and 45% of the methionine is present as the sulphoxide in the nuclear proteins. The levels of other amino acids appeared to remain constant. 4. Similar, but smaller, changes were found in the cortical proteins in advanced nuclear cataractous lenses, suggesting that the oxidation spreads from the nucleus to the cortex. 5. These changes were discussed with regard to current views on cataract formation and it was concluded that they are probably the result of simple oxidation of the proteins with O2 or H2O2.

On the structure of alpha-crystallin: construction of hybrid molecules and homopolymers.

The alpha A2 and alpha B2 subunits of bovine alpha-crystallin were purified by chromatofocussing in urea and assembled into homopolymers. Light-scattering measurements indicated their molecular masses were 360 and 420 kDa. The alpha A2 and alpha B2 polypeptides were also used to construct a series of hybrid molecules with alpha A/alpha B ratios ranging from 7:1 to 1:7. Sedimentation velocity analyses, isoelectric focussing under non-deaggregating conditions, circular dichroism spectroscopy and immunochemical analysis indicated that all of the subunits had copolymerized to alpha-crystallin-like aggregates with complete regeneration of the native structure. The polymers could be distinguished on the basis of their differing affinities for the antiserum. This was directly related to the proportion of alpha A2 subunits in each polymer. It was concluded that the alpha A2 and alpha B2 subunits are structurally equivalent and occupy equivalent site in the alpha-crystallin aggregates. It was also concluded that a micellar-like quaternary structure was consistent with most previous observations on the protein.

The effect of phosphorylation on the structure of alpha-crystallin.

Homopolymers were constructed from the highly purified phosphorylated (A1 and B1) and non-phosphorylated (A2 and B2) polypeptides of alpha-crystallin. These were examined using electron microscopy, light scattering, fluorescence spectroscopy and sulphydryl probing to determine the effects of phosphorylation on the structure of alpha-crystallin. Each reconstituted aggregate consisted of uniform particles with circular cross-sections and diameters of 9.3-9.5 nm. Some of these were associated in chain-like structures. The molecular mass of the homopolymers varied from 360-507 kDa; for a single particle, it was estimated to be 216 +/- 10 kDa. Tryptophan residues in the alpha B2 homopolymers were more accessible to the solvent and to quenchers than those in alpha A2. Phosphorylation increased this accessibility in the alpha A homopolymer but decreased it in alpha B. This decrease could be attributed to phosphorylation of the serine adjacent to tryptophan 60 in the B chain. The kinetics of the reaction with DTNB indicated that the single cysteine in the A chain was buried in the alpha A2 homopolymer (k1' = 0.013 min-1) but more accessible in alpha A1 (k1' = 0.046 min-1). It was concluded that phosphorylation significantly alters the conformation of the alpha A subunits but probably has little effect on the B subunits. The alterations do not affect the ability of the subunits to associate into alpha-crystallin-like particles.

Studies on the location of aromatic amino acids in alpha-crystallin.

The locations of tryptophan residues in alpha-crystallin and homopolymers constructed from the alpha A- and alpha B-chains were examined by comparing their fluorescence emission properties and their accessibilities to quenchers. Two classes of tryptophan could be distinguished on the basis of differences in their spectral characteristics, fluorescence decay lifetimes, quenching with acrylamide and exposure by increasing concentrations of urea. Polarization measurements indicated that the tryptophan residues were associated with flexible segments of the polypeptide chains. The two classes could be assigned, one to Trp-9 (in both A- and B-chains) which is in an hydrophobic environment, and one to Trp-60 (B-chain) which appeared to be nearer the surface of the aggregate. No evidence was found for residues inaccessible to the quenchers. An apparent partition coefficient of 40 was obtained for the association of acrylamide with the protein. From temperature-dependence studies, it was concluded that there was a significant energy barrier to the penetration of acrylamide into the protein matrix (Ea = 5.8 kcal/mol) and that entry of the quencher was through channels produced by temporary disruption of the matrix (delta s = 1.5 eu). The phenolic side-chains of tyrosine residues in several different alpha-crystallins were found to ionize with pK values above pH 11, indicative of residues highly shielded from the solvent. Comparisons of polypeptide sequences, together with tyrosine fluorescence emission data and the pK values, permitted a tentative assignment of residue locations. All of the data are consistent with a possible micelle-like structure for alpha-crystallin but not with a layered structure.

Specific dissociation of alpha B subunits from alpha-crystallin.

Exposure of bovine alpha-crystallin to 0.1 M glycine at pH 7 decreases the average molar mass of the protein from 700 to 420 kDa. When the pH is lowered to 2.5, in the same buffer, the alpha B chains specifically dissociate from the aggregates, leaving a particle of 290 kDa containing only alpha A chains. The decrease in the molar mass corresponds to the mass of the alpha B chains in the original aggregate. The pH-dependent dissociation is fully reversible. Similar changes were observed with rat and kangaroo alpha-crystallins but the dogfish protein was not affected. Sedimentation velocity analyses and fluorescence spectroscopy yielded a pK, for the dissociation, of 3.7 for alpha-crystallin and 4.0 for a homopolymer constructed from purified alpha B2 polypeptides. An alpha A2 homopolymer was virtually unaffected by the lowering of pH. The products from the dissociation were isolated and their properties studied by sedimentation analysis and acrylamide quenching of tryptophan fluorescence. The alpha B chains were found to be completely denatured, whereas the structure of the alpha A chains, in the 290 kDa, particle, were only slightly altered. Comparisons of the sequences of the various proteins examined suggested that decreased ionization of aspartic acid 127 in the alpha B chain was responsible for the specific dissociation of this polypeptide.

The location of sulphydryl groups in alpha-crystallin.

The microenvironments of the sulphydryl groups in the multimeric protein, alpha-crystallin, were studied by examining: the rate of the reaction of the groups with DTNB; the effect of increasing urea concentrations on their accessibilities; and the quenching of a fluorescent probe. In foetal bovine alpha-crystallin (1 SH/alpha A subunit) both kinetic and quenching studies indicated that over 90% of the sulphydryl groups fell into a single buried class; the remainder was exposed. In the human protein (2 SH/alpha A subunit), half of the groups were buried and the other half exposed. Accessible sulphydryl groups increased gradually as the urea concentration was increased, with complete exposure at about 4.0 M. Sedimentation velocity analyses revealed that no significant dissociation of the aggregates into subunits occurred below 3.5 M urea, at which point over 80% of the sulphydryl groups were exposed. An age-dependent increase (3-35%) was found in the proportion of exposed sulphydryl groups in bovine alpha-crystallin and a decrease in the urea concentration required to expose the remainder. It was concluded that the single cysteine is buried in the newly synthesized protein, but becomes solvent-exposed as a result of age-related conformational changes. Our observations are consistent with a quaternary structure in which all alpha A subunits occupy equivalent sites.

Probing the microenvironments of tryptophan residues in the monomeric crystallins of the bovine lens.

Tryptophan microenvironments have been examined in bovine beta s-, gamma II-, gamma IIIa-, gamma IIIb-, gamma IVa- and gamma IVb-crystallins by fluorescence methods. The proteins could be divided into two groups on the basis of the accessibilities of their tryptophan residues. The first group, comprising beta s, gamma II and gamma IIIb, appeared to have a compact structure with none of the tryptophans accessible to KI and only moderately so to acrylamide. By contrast in gamma IIIa, gamma IVa and gamma Vb, all tryptophans were readily accessible to acrylamide and 70% of the fluorescence could be quenched with KI. Spectral analysis, before and after quenching, time-resolved spectroscopy and simulations of the quenching curves suggested that gamma IIIa, gamma IVa and gamma IVb contain two classes of tryptophan residues. One class (tau 0 = 0.52 ns, fa = 0.3, lambda max = 324 nm) which was completely inaccessible to KI and relatively inaccessible to acrylamide (Ksv = 0.25 M-1), was assigned to the topologically equivalent residues in positions 42 and 131. The other class (tau 0 = 2.1-3.4 ns, fa = 0.7, lambda max = 330 nm) was accessible to both quenchers (Ksv = 5.00-5.15 M-1 and 2.47-2.60 M-1, for acrylamide and KI, respectively) and corresponded to the tryptophan residues in positions 68 and 157. The same classes may be present in the other low molecular weight proteins (tau 0 = 0.47-0.55 and 1.55-1.74) but the lower emission and low accessibilities to quenchers prevented their distinction and suggested that these proteins had more compact structures.

Protein synthesis and secretion by cultured retinal pigment epithelia.

Protein synthesis and secretion by post-natal sheep and calf retinal pigment epithelial (RPE) cells was investigated following labelling of choroidal pieces, isolated RPE cells and RPE cells in tissue culture with L-[U-14C] leucine. We show that RPE cells secrete a specific set of proteins that includes retinol binding protein (RBP) and transthyretin (TTR), which are both involved in retinol transport in blood. Using a two-chambered culture system we show that protein secretion by the post-natal RPE cells occurs predominantly across the apical pole of the cells, i.e., across the surface of the cells which, in vivo, faces the retina. In agreement with results of others using foetal RPE cells (Ong, D.E., Davis, J.T., O'Day, W.T. and Bok, D. (1994) Biochemistry 33, 1835-1842) we show that RBP and, to a lesser extent, TTR are also secreted predominantly across the apical pole of the cell. We have developed a cell culture model for the RPE that may be used as an in vitro model for studying transport across the blood-retinal barrier.

A possible structure for alpha-crystallin.

alpha-Crystallin, the major protein of the mammalian eye lens, is found in vivo as a multimeric aggregate composed of two closely related subunits whose molar ratio is widely variable from species to species. Attempts to determine the arrangement of the subunits within the aggregate, or even to determine the size of the aggregate and the number of subunits composing it, have not resulted in general agreement. Because of the variability in alpha-crystallin particle size, the apparent dependence of this parameter on certain environmental factors (e.g. temperature), the absence of a specific requirement for either alpha-crystallin isoform in aggregation, and the sharp division in the amino acid sequence between a strong hydrophobic region and a sharply hydrophilic one, it is suggested that the alpha-crystallin aggregate has the properties of a protein micelle. This hypothesis is consistent with what is known of the alpha-crystallin molecule and aggregate, and can be tested experimentally. If this hypothesis is shown to be true, then alpha-crystallin will be the first example of a naturally occurring protein micelle.

Binding of dexamethasone by alpha-crystallin.

PURPOSE: Long-term steroid therapy is a known risk factor for the development of posterior subcapsular cataract. Previous work in this laboratory has found soluble lens proteins to bind dexamethasone, but this binding is not due to a glucocorticoid receptor. This study was undertaken to identify the soluble protein or proteins involved in lens glucocorticoid binding. METHODS: Bovine lens extract was incubated with 5.2 x 10(-)(8) M [(3)H]-dexamethasone for 3 hours, and the distribution of label assessed in the soluble and insoluble fractions after centrifugation. Soluble lens extract was fractionated using gel permeation chromatography to isolate and identify proteins involved in the binding. Total lens proteins, high-molecular-weight proteins, or alpha-crystallin were exposed to dexamethasone and the protein bound steroid measured after separation of free and bound ligand on a gel chromatography column. Scatchard analysis was used to determine dexamethasone-binding parameters. Sequence comparisons between bovine alphaA- and alphaB-crystallins and glucocorticoid-binding proteins were performed using a sequence-alignment program. RESULTS: Of the total dexamethasone bound in lens extract, soluble proteins were found to account for 52%. The majority of the soluble protein-bound dexamethasone coeluted with the high-molecular-weight proteins that consisted mainly of alpha-crystallin. Binding studies with isolated proteins showed that alpha-crystallin accounted for more than 98% of total soluble dexamethasone binding in the lens. Scatchard analysis of steroid binding showed it to be a nonspecific partitioning event. Sequence comparisons between alphaA- and alphaB-crystallins and various glucocorticoid-binding proteins showed the lens proteins to have three regions of sequence homology with yeast corticosteroid-binding protein. CONCLUSIONS: alpha-Crystallin is the principal soluble glucocorticoid binding protein in the lens. The steroid association is described by nonspecific partitioning and may be related to the unique structural characteristics of the protein. The nonspecific association with alpha-crystallin is not thought to be functional; however, it may aid in the increased covalent steroid modification observed for this protein.

Expression of Crystallins, Pax6, Filensin, CP49, MIP, and MP20 in lens-derived cell lines.

PURPOSE: Cell lines are the systems of choice to analyze cellular functions related to the particular organ system. For lens research, three cell lines are widely used: N/N1003A (derived from rabbit lenses), alpha TN4, and NKR-11 (both of murine origin). The aim of the current study was to characterize these particular cell lines with respect to their expression of genes that are considered to be lens specific or expressed preferentially in the lens, such as crystallins, Pax6, Filensin, CP49, MIP, and MP20. METHODS: alpha A- and alpha B-crystallin cDNA from rabbit lenses were sequenced. The expression of various genes was analyzed by reverse transcription-polymerase chain reaction using specific primers and mRNA from three lens-derived cell lines. For control, the expression of the selected genes was compared in nonlenticular tissues of mouse as well as in non-lens-derived murine cell lines (EF43, NIH-3T3, and L929). RESULTS: None of the transcripts for beta B2-crystallin, gamma-crystallins, MIP, MP20, filensin, and CP49 could be detected in the lens-derived cell lines. Transcripts for alpha A-crystallin were amplified in alpha TN4, but not in N/N1003A and NKR-11 cells. Pax6, a master control gene of eye development, is expressed in all three lens-derived cell lines and, additionally, in cell lines of neuronal origin, but not in corneal endothelial cells and in the currently used control cell lines. CONCLUSIONS: Three cell lines of lenticular origin were tested for expression of genes that were found abundantly in the lens. The observed expression of Pax6 in all lens-derived cell lines allows their use in the analysis of corresponding signal chains.

The refractive increments of bovine alpha-, beta-, and gamma-crystallins.

The refractive increments have been determined for purified bovine alpha-, beta- and gamma-crystallins, using three different light sources. The values obtained were used to predict the refractive index gradient in the rat lens. Excellent agreement was obtained between the predicted and observed gradients.

Gradient refractive index of the crystalline lens of the Black Oreo Dory (Allocyttus Niger): comparison of magnetic resonance imaging (MRI) and laser ray-trace methods.

The gradient refractive index of the crystalline lens in the Black Oreo Dory (Allocyttus Niger) was determined using two methods; an optimisation program based on finite ray-tracing and the path of laser beams through the lens, and magnetic resonance imaging (MRI) and the linear relationship between refractive index and nuclear transverse relaxation rates. The methods showed good agreement in the cortical zone of the lens, but the lack of free water in the core of the lens made MRI measurement impossible in this region. The laser-optimisation method gave mean values of 1.368 and 1.543 for the surface and core refractive indices respectively, with a radial distribution for the gradient refractive index given by n(r)=1.543-0.121r2-0.033r4-0.021r6.

Isolation of alpha-crystallin subunits by gel filtration.

In the presence of 0.1 M glycine, pH 2.5, alpha-crystallin dissociates into aggregates containing only alpha A chains plus monomeric alpha B chains. Advantage has been taken of this to develop a method for the purification of the alpha A and alpha B chains by gel filtration on Sephadex G-75. This method gives high yields of purified subunits and avoids the risk of carbamylation associated with other methods which use high concentrations of urea.

On the structure of alpha-crystallin: the minimum molecular weight.

alpha-crystallin can be isolated in two forms depending on the temperature at which the lens is extracted. At 4 degrees C, alpha c-crystallin is obtained while at 37 degrees C, a smaller molecule, alpha m-crystallin can be isolated. The apparent molecular weight of bovine foetal alpha m- and alpha c-crystallins were determined in 5 different ways using sedimentation velocity, sedimentation equilibrium and intensity fluctuation spectroscopy analyses and the experimentally determined diffusion coefficients, intrinsic viscosity and partial specific volume. Values ranged from 291,000 to 369,000 for alpha m and from 604,000 to 760,000 for alpha c due to the differential effects of the protein's polydispersity on the different methods. Subfractionation of the protein by gel filtration yielded much less polydisperse minimum species populations with molecular weights of 280,000 and 529,000 for alpha m and alpha c respectively. It was concluded that alpha-crystallin is probably synthesized as a symmetrical dodecamer and that the polydispersity of most preparations can be attributed to age-related modification in vivo as well as in vitro supra-aggregation due to variations in experimental conditions.

Protein distribution patterns in concentric layers from single bovine lenses: changes with development and ageing.

Protein distribution patterns were determined in concentric layers removed from 24 bovine lenses ranging in age from about 6 months before birth to 180 months post-natal. It was possible to distinguish alterations in protein synthesis patterns during development and changes due to ageing, i.e., prolonged existence of the proteins. It was found that alpha-crystallin represents a constant 50% of the proteins synthesized by the fibre cells throughout life. However, the protein becomes progressively less soluble with increasing age. Beta-crystallin synthesis increases from 30% of the total proteins during prenatal development to around 40% in post-natal fibre cells. This increase is due to increased production of the beta-crystallin. In old tissues, beta H-crystallin is converted to a high molecular weight from (HMW beta) gamma-crystallins account for 22% of the proteins synthesized in the earliest prenatal fibre cells. This level decreases rapidly through prenatal development until they represent about 4% of the total at birth. Beta S-crystallin synthesis commences around this time and in the post-natal fibre cells is essentially the only low molecular weight protein. The possible significance of some of these changes is discussed with regard to the functional requirements of the lens.

Electron microscopy of native and reconstituted alpha crystallin aggregates.

The size and shape of native alpha crystallin aggregates extracted at either 4 degrees C (alpha c-crystallin) or 37 degrees C (alpha m-crystallin), were compared with each other, as well as with aggregates reconstituted from either pure alpha A or alpha B subunits using electron microscopy. The alpha B aggregates were the most uniform in size (about 9 nm in diameter) and the best stained. alpha A particles were about the same size as the alpha B, but the population distribution was broader and some indication of interparticle association was observed. alpha c particles exhibited a bimodal distribution, with one peak greater than the reconstituted particles and the other about the same size; alpha m was smaller than the reconstituted structures.

Conformational changes in soluble lens proteins during the development of senile nuclear cataract.

Soluble crystallins were isolated, by gel filtration, from the nucleus and cortex of types I-IV cataractous lenses and neonatal lenses. Extinction coefficients were determined and used to calculate the proportion of the crystallins. alpha Crystallins were the major proteins in the cortex of the cataractous lens whereas beta crystallins predominate in the nucleus and in the neonatal lens. Electrophoretic and immunological data, indicated that there was very little soluble gamma crystallin in the cataractous tissue. Highly specific competitive radioimmunoassays were used to estimate the crystallin contents of the various extracts. Values obtained for the neonatal extracts agreed closely with those from gel filtration but, in general, the values found for the cataractous proteins were low. The immunological reactivity of beta crystallin decreased progressively with the development of nuclear colour whereas alpha crystallin reactivity decreased sharply with the onset of nuclear cataract (type II lens) and then partially recovered. No significant alterations were found in the micro-environments of tryptophan residues in any of the proteins indicating that there are no gross conformational changes in the soluble proteins during cataract development. It would appear that the losses of immunochemical reactivity are due to chemical and/or conformational alterations which are restricted to the surface of the protein molecules.