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.

Dissociation is not required for alpha-crystallin's chaperone function.

Bovine alpha-crystallin was crosslinked with glutaraldehyde under conditions designed to minimise intermolecular reactions. The crosslinked protein was too large to enter SDS polyacrylamide gels but HPLC-gel permeation chromatography revealed that the Stoke's radii of the native and crosslinked proteins were very similar. These observations indicate that only intramolecular crosslinks had formed and that the crosslinked protein could not dissociate to smaller species. The crosslinked alpha-crystallin was able to inhibit the thermally-induced precipitation of beta-crystallin and appeared to be more effective than the native protein under the same conditions. It is concluded that the chaperone activity of alpha-crystallin is a surface phenomenon and dissociation into smaller species is not required.

The frequency of the canine leukocyte adhesion deficiency (CLAD) allele within the Irish Setter population of Australia.

OBJECTIVE: To determine the frequency of the 107G-->C canine leukocyte adhesion deficiency (CLAD) mutation in Irish Setters from the Australian breeding population. METHOD: Genomic DNA was isolated from 87 Irish Setter blood samples and a region of the beta-2 integrin gene (ITGB2), encompassing the mutation, was amplified using real-time Polymerase Chain Reaction (PCR). Two fluorescently labelled probes were hybridised to the fragment, and fluorescence resonance energy transfer (FRET) was used to detect the 107G-->C mutation responsible for CLAD. RESULTS: Three new heterozygotes were identified among 87 healthy Irish Setters from Australia. All originated from a litter sired by a known heterozygote. A total of seven heterozygotes have now been identified in 92 dogs (7.6%), representing over 90% of all major breeding stock in five Australian states. Two of the heterozygotes were recently imported adult dogs and the others were their offspring. CONCLUSIONS: The frequency of the 107C allele in the Australian population of Irish Setters is lower than that in Europe. Selective breeding programs should be adopted to eliminate the mutant allele presently in two breeding lines.

Is there a glucocorticoid receptor in the bovine lens?

Prolonged glucocorticoid therapy is a risk factor for cataract development. The mechanism remains unknown. If cataract results from the direct effect of steroids on lens function, a glucocorticoid receptor is required. In order to determine whether such a receptor was present in the bovine lens, metabolic and steroid binding experiments were undertaken. Cultured bovine lens epithelial cells were exposed to 10(- 4)and 10(-8) M dexamethasone or prednisolone and the uptake and incorporation of(14)C leucine,(14)C glucose and(3)H thymidine, examined. Neither glucocorticoid affected cell protein synthesis or glucose uptake. Both dexamethasone concentrations and the lower concentration of prednisolone had no effect on thymidine uptake or incorporation, however, the 10(-4) M prednisolone exposure reduced these by 15 +/- 5%. This regulation is thought to be due to membrane fluidity changes and not the action of the glucocorticoid receptor. As the glucocorticoid receptor is very heat labile in vitro, the effects of increasing temperature on dexamethasone binding by proteins from lens epithelium, lens nucleus and liver were examined. At 0 degree C, lens epithelial extract bound nine-fold more dexamethasone than liver extract. After exposure to 37 degrees C, liver binding decreased by 66% whereas that for lens epithelium increased by 18%. For both lens extracts, steroid binding increased with temperature up to 50 degrees C. Scatchard analysis of the steroid binding kinetics showed there to be no high affinity sites in lens epithelial extract, with the binding best described as a non-specific partitioning event. Western blotting with a specific glucocorticoid receptor antibody revealed protein bands of approximately 94 and 79 kDa in liver, which is known to contain significant levels of receptor. No immunoreactivity was observed for lens epithelial extract. Therefore, within the limits of detection, these results suggest the bovine lens does not contain a glucocorticoid receptor. This raises questions about the validity of receptor-mediated mechanisms proposed for cataract development.

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.

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.

Genetic screening for progressive retinal atrophy in the Australian population of Irish Setters.

OBJECTIVE: To determine whether the rcd-1 mutation causing progressive retinal atrophy (PRA) in Irish Setters is in the Australian breeding population. METHOD: DNA samples were tested for the mutation using the Polymerase Chain Reaction and specific primer nucleotides to amplify the phosphodiesterase gene followed by restriction enzyme cleavage and fragment size determination. RESULTS: No mutant alleles were found in 38 Irish Setters, representing over 80% of all major breeding stock in five Australian states. CONCLUSIONS: It is likely that the Australian population of Irish Setters is free of the rcd-1 form of PRA.

alpha-Crystallin polymers and polymerization: the view from down under.

Several models have been proposed for the quaternary structure of alpha-crystallin. Some suggest the subunits are arranged in concentric shells. Others propose that the subunits are in a micelle-like arrangement. However, none is able to satisfactorily account for all observations on the protein and the quaternary structure of alpha-crystallin remains to be established. In this review, factors contributing to the assembly and polymerization are examined in order to evaluate the different models. Consideration of the variations in particle size and molecular weight under different conditions leads to the conclusion that alpha-crystallin cannot be a micelle or a layered structure. Instead, it is suggested that the protein may be assembled from a 'monomeric' unit comprising eight subunits arranged in two tetramers with cyclic symmetry. The octameric unit is proposed to be disc-like particle with a diameter of 9.5 nm and a height of 3 nm. The larger particles, chains and sheet-like structures commonly observed are assembled from the octamers. Structural predictions indicate that the polypeptide may be folded into three independent domains which have different roles in the structural organization and functions of the protein. It is suggested that the tetramers are stabilized through interactions involving the second domain (residues 64-104) while assembly into the octamers and higher polymers requires hydrophobic interactions involving the N-terminal domain. Deletion of parts of this domain by site directed mutagenesis revealed that residues 46-63 play a critical role in the assembly. Current research aims to identify the specific amino acids involved.

The effect of light deprivation on the mouse lens.

This work was undertaken to test the hypothesis that first exposure of the eye to light is responsible for the changes in lens protein expression patterns observed around the time of birth. The effect of light deprivation on lens properties was examined in Balb c mice which were bred, reared and maintained in complete darkness for up to 7 months. Data were collected on body weight, lens weight, lens protein contents and crystallin distributions. The data were compared with those obtained from age matched mice maintained in natural light/dark conditions. No significant differences were observed in body weight between animals maintained in the light and dark. However, animals kept in the dark had significantly smaller lenses. After 6 months in the dark, the lens represented 0.02% of body weight compared with 0.031% in the light reared animals (P < 0.001). Lens protein concentration, insoluble protein contents and crystallin synthesis patterns were indistinguishable for the two groups of animals. It is concluded that light stimulation of the eye is required for optimal lens growth but does not affect the production of specific crystallins.

Binding of 1-anilinonaphthalene-8-sulfonic acid to alpha-crystallin.

alpha-Crystallin was found to exhibit a time-dependent uptake of the hydrophobic probe, 1-anilinonaphthalene-8-sulfonic acid (ANS), similar to that typically observed with lipid membranes. Analysis of the interaction of ANS with alpha-crystallin revealed two types of interactive processes, partitioning and binding. The predominant process involved partitioning, with a coefficient of 300 M-1. The binding component had the following characteristics: 1 binding site/24 subunits and a Kd of about 9 microM. The binding was unaffected by the number of subunits used in the assembly of the alpha-aggregate, since both the alpha m- and alpha c-forms had similar binding characteristics. No discernible differences were observed in the binding of ANS to homopolymers of alpha A and alpha B subunits, suggesting that the hydrophobic sites to which ANS bound were similar in both the A and B subunits. The majority of the fluorescence was lost when the protein was incubated in 3 M urea, a concentration of denaturant where the protein is still intact, suggesting that the ANS binding sites are located near the surface of the protein. The decrease was attributed to a decrease in the quantum yield of the bound dye.

The Cryner element in the murine gamma-crystallin promoters interacts with lens proteins.

Based upon DNA sequence analysis of the promoters from six gamma-crystallin genes (cryga-->crygf) a 36-bp DNA fragment was defined as 'Cryner' (cryg nested repeat). The presence of these repeats made this structure a candidate for DNA-protein interaction. The present experiments demonstrate interactions of lens proteins with the Cryner element from murine cryga, crygb, crygd and cryge. Additionally, DNA covering the sequence of about 30 nt between Cryner and the TATA-box of the murine crygb exhibits sequence-specific interactions with the bovine alpha-crystallin-containing fraction. The results confirm the hypothesis that the Cryner element is able to interact with lens proteins. It is noteworthy that this interaction is specific for the template strand of the DNA. The present model includes the possibility of sequence-dependent conformational changes leading to various DNA-protein complexes.

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 effect of concentration on the structure of alpha-crystallin.

Several models have been proposed for arrangement of the subunits in alpha-crystallin. These include the contrasting proposals that subunits are arranged in three layers and that subunits assemble into micelle-like structures. The validity of the micelle model was investigated by examining the effects of variations in protein concentration on the surface tension, conductivity, molecular weight and conformation of alpha-crystallin. The data were compared with those obtained for bovine serum albumin (BSA) and sodium dodecyl sulphate (SDS). Measurements of surface tension were conducted in the range, 10 micrograms ml-1 to 130 mg ml-1, in low and high ionic strength buffers. An apparent point of inflection, independent of ionic strength, was seen in alpha-crystallin's surface tension at around 1.9 mg ml-1 (95 microM). The surface tension did not plateau beyond this point, as is the case with surfactants, but continued to decrease up to 130 mg ml-1. BSA exhibited similar surface tension properties with an apparent inflection at 0.9 mg ml-1 (13 microM). The conductivity of alpha-crystallin and BSA solutions increased smoothly with no sign of any transition up to 96 mg ml-1 and 60 mg ml-1, respectively. In contrast, SDS showed a clear transition in this property at the concentration corresponding to its CMC. The aggregation state of the alpha-crystallin aggregates was examined by comparing molecular masses and Stokes radii. The size of the protein remained uniform over a wide concentration range and was unaffected by variations in ionic strength. Protein conformation, which was monitored by examining the microenvironment of tryptophan residues, was also found to be independent of protein concentration. It is concluded that over the concentration range that was investigated, alpha-crystallin does not exhibit any of the properties associated with classical micelles formed from small amphiphilic molecules.

A sheet-like form of alpha-crystallin.

Complexes containing alpha-crystallin were isolated from crosslinked lens extract using an affinity column constructed with monoclonal antibodies specific for alpha-crystallin. The affinity-purified protein was compared with alpha-crystallins before and after crosslinking. Electron microscopy revealed sheet-like structures in the cross-linked protein from the lens extract compared with spherical structures for the others. Studies on the amino acid composition, tryptophan microenvironments and the interaction with a monoclonal antibody revealed that the complexes consist almost entirely of alpha-crystallin. These results indicate that under certain conditions, alpha-crystallin subunits can adopt a sheet-like form.

The refractive index and protein distribution in the blue eye trevally lens.

BACKGROUND: The relationship between structure (crystallin distribution) and function (refractive index) in the lens is not understood and can be studied by comparing biochemical and optical properties. Such a comparison has been made using a blue eyed trevally lens. METHODS: The optical parameter of refractive index distribution was determined using a nondestructive ray tracing technique. The distributions of the various classes of proteins in the lens were determined by dissolving lenses in concentric layers and using biochemical protein assay. HPLC and SDS-PAGE electrophoresis were used to investigate the proportion of proteins in each layer. RESULTS: The refractive index distribution, from center to edge, follows a second order polynomial. The proteins do not vary in their proportions over most of the lens; only in the inner-most regions is there a rapid increase in insoluble protein and a concomitant decrease in the soluble protein classes. The smallest proteins (gamma crystallins) become insoluble later than the alpha- and beta-crystallins. CONCLUSIONS: There are no similarities in the distributions of any of the protein classes to that of the refractive index in the fish lens. This result indicates that a quantitative relationship cannot be derived by comparing protein to refractive index distributions. However, the findings are consistent with those made in other species: a high content of gamma-crystallins is always found in lenses which have steep refractive index gradients and high index magnitudes.

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.

Ageing of glutathione reductase in the lens.

The distribution of glutathione reductase activity in concentric layers from the lens has been determined as a function of age for 16 species. Primate lenses have almost ten times the level of glutathione reductase found in other species. Comparison with the activity of hexokinase revealed that this is not due to a higher overall rate of metabolism in these lenses. By contrast, the higher activity found in bird and fish lenses reflects a higher metabolic activity in these tissues. In all species, a gradient of activity was observed with the highest specific activity in the outermost cortical fibres, decreasing to virtually no activity in the inner parts of the tissue. No alterations were found in this gradient with increasing age, other than an increase in the amount of nuclear tissue essentially devoid of activity. The maximum activity in the outer cortical fibres was the same, regardless of the age of the lens. The time taken, in different species, for the specific activity to decrease by half, was estimated from the rate of protein accumulation. This time was found to vary from a few days to several years, indicating that the decrease in activity is not due to ageing but rather, it is related to the maturation of fibre cells. These observations are discussed in terms of current concepts of lens ageing and cataract formation.

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.