July 2001
Volume 42, Issue 8
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Lens  |   July 2001
Binding of Dexamethasone by α-Crystallin
Author Affiliations
  • Andrew Ian Jobling
    From the National Vision Research Institute of Australia, Carlton, Victoria, Australia.
  • Arthur Stevens
    From the National Vision Research Institute of Australia, Carlton, Victoria, Australia.
  • Robert Cornelis Augusteyn
    From the National Vision Research Institute of Australia, Carlton, Victoria, Australia.
Investigative Ophthalmology & Visual Science July 2001, Vol.42, 1829-1832. doi:
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      Andrew Ian Jobling, Arthur Stevens, Robert Cornelis Augusteyn; Binding of Dexamethasone by α-Crystallin. Invest. Ophthalmol. Vis. Sci. 2001;42(8):1829-1832.

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Abstract

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 × 10 8 M [3H]-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 α-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 αA- andα B-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 α-crystallin. Binding studies with isolated proteins showed that α-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 αA- and αB-crystallins and various glucocorticoid-binding proteins showed the lens proteins to have three regions of sequence homology with yeast corticosteroid-binding protein.

conclusions. α-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 withα -crystallin is not thought to be functional; however, it may aid in the increased covalent steroid modification observed for this protein.

The prolonged use of systemic, 1 topical, 2 or inhaled 3 glucocorticoids is known to be a high risk factor in the formation of posterior subcapsular cataract. Several earlier reports implicated glucocorticoid receptor–mediated regulation of lens metabolism. 4 5 6 However, recent work from this laboratory has shown that, whereas soluble lens proteins bind steroid, the bovine lens does not contain a classic cytosolic glucocorticoid receptor. 7  
Apart from the cytosolic receptor, several soluble as well as membrane-bound, steroid-binding proteins have been described. 8 9 10 Some have been observed in the lens. In 1973, Ono et al. 11 described a cortisol-binding protein found in the β-crystallin fraction of rat lens extract. The protein was implicated in cataract formation, because its level was found to decrease in cataractous human lenses. More recently, Cenedella et al. 12 have reported that bovine lens epithelial membranes contain a 28-kDa protein capable of binding progesterone with high affinity. 
Lens proteins are also known to covalently bind steroids through their lysine residues. 13 Dickerson et al. 14 observed a number of steroids, including progesterone, to bind purifiedβ -crystallin. One of the more potent glucocorticoids, dexamethasone, has also been reported to preferentially bindα -crystallin. 15 Although a small amount of lens-soluble steroid binding can be attributed to covalent interactions, the majority of the binding is not of this type. 7  
In the mammalian system, the soluble steroid-binding proteins are principally found in serum and can be classified into three groups, the sex hormone–binding globulin (SHBG), the corticosteroid-binding globulin (CBG), and serum albumin. 16 These form noncovalent complexes with steroids through a combination of hydrophobic and hydrophilic forces. This binding, together with high dissociation rates, 10 generates a readily dissociable complex, allowing instantaneous control of circulating steroid levels. Because protein-associated steroid is known to be functionally inactive and specific cell membrane–binding sites have been reported, 17 these serum proteins act as important biological mediators. 18 The soluble lens steroid–binding protein may function in the same way. 
The purpose of the present study was to identify the soluble protein responsible for glucocorticoid binding in lens extract and to characterize its steroid-binding properties. 
Materials and Methods
Bovine eyes were obtained from local abattoirs and processed within 2 hours of death. All subsequent procedures were performed at 4°C. The lenses were removed, decapsulated, and homogenized in PBSA (7.2 mM Na2HPO4, 2.8 mM NaH2PO4, 0.15 M NaCl, 0.1% NaN3 [pH 7.2]). The extract was centrifuged at 13,300g (J2–21 centrifuge; Beckman Coulter, Fullerton, Ca) for 30 minutes and the supernatant stored at 4°C. Protein concentrations were estimated with the method of Lowry et al. 19  
Protein Purification
Soluble lens proteins were separated in PBSA by gel permeation chromatography 17 (GPC; 800 HRLC; Bio-Rad, Richmond, CA; incorporating Zorbax G450 and G250 Bioseries columns; DuPont NEN, Boston, MA, connected in series). A flow rate of 1 ml/min was used, and the absorbance was measured at 280 nm (A280; 1706 UV/VIS; Bio-Rad). The polypeptide content of the isolated protein fractions was examined using SDS-PAGE, as described previously. 20  
An α-crystallin monoclonal antibody Sepharose-affinity gel was used to purify α-crystallin. The production of the column and the method of α-crystallin isolation were as described by Stevens and Augusteyn. 21 The eluted protein was renatured by dialysis against Tris buffer (50 mM Tris, 2 mM EDTA, 0.02% NaN3 [pH 8.0]) containing 7 M urea and subsequently against the Tris buffer alone. The protein was further dialyzed against PBSA and stored at 4°C. 
Dexamethasone Binding
To identify the proteins interacting with dexamethasone, lens extract or soluble lens proteins were incubated with 5.2 × 10 8 M[ 6,7-3H]- dexamethasone (39.2 Ci/mmol; DuPont NEN) for 3 hours at 20°C. For whole lens extracts, the soluble and insoluble fractions were separated by centrifugation and the distribution of radioactivity determined (1215 Rackbeta II or model 1409 liquid scintillation counter; Wallac, PerkinElmer Life Science, Boston, MA). Correction for contamination of the insoluble fraction with the soluble phase was made by reference to the distribution of 32Pi (9000 Ci/mmol; DuPont NEN) added before centrifugation. The soluble proteins were separated by GPC as described, individual fractions were collected, and the associated radioactivity was determined. 
All other dexamethasone-binding studies were performed essentially as follows. Proteins, ranging in concentration from 0.1 to 50 mg/ml, were incubated at 20°C with 5.2 × 10 8 M[ 6,7-3H]-dexamethasone for 3 hours, after which the protein-bound and free steroid were separated on a 1 × 10-cm chromatography column (Sephadex G-25; Amersham Pharmacia Biotech, Piscataway, NJ) equilibrated with PBSA. 
Determination of steroid-binding parameters using Scatchard analysis involved the addition of 5 to 315 × 10 9 M[ 6,7-3H]-dexamethasone to a 2 mg/ml solution ofα -crystallin and incubation for 3 hours at 20°C. Samples were taken before separation of the bound and free steroid to estimate total steroid concentration and allow the calculation of free steroid. The amount of bound dexamethasone was calculated per mole of α-crystallin subunit, using an average subunit molecular weight of 20,000. 
Sequence Comparison
The sequences of bovine αA- and αB-crystallins were compared with those of known glucocorticoid-binding proteins. Sequence information was obtained from the Swiss-Prot database (Swiss Institute of Bioinformatics, Geneva, Switzerland; available in the public domain to academic users [licensing fee charged to commercial users] at http//:www.expasy.org), and alignments were performed using the a multiple sequence-alignment program (Clustal W, ver. 1.7; SGI, Mountain View, CA). 
Results
Identification of Steroid Binding Protein
Total lens extract, including the insoluble fraction, was incubated with dexamethasone for 3 hours at 20°C. Approximately equal amounts of dexamethasone, 2.7% and 2.5% of that added, were found to be associated with the soluble and insoluble fractions, respectively. The steroid in the insoluble fraction is most likely associated with the steroid-binding membrane protein previously described by Cenedella et al. 12 However, the identity of the soluble proteins interacting with dexamethasone is unclear. At 20°C, the soluble cortisol-binding protein 11 and glucocorticoid receptor 22 are inactivated, leaving only nonspecific components of the interaction. 
To identify the interacting species, the soluble proteins were fractionated using GPC. As can be seen in Figure 1 , substantial radiolabel is associated with the high-molecular-weight (HMW; α-crystallin) fraction and only small amounts (<5% of HMW) with the medium-molecular-weight (MMW; β-crystallin) fraction. The radioactivity peak observed at approximately 29.5 minutes and apparently associated with the low-medium-weight (LMW; γ-crystallin) proteins, is due to free dexamethasone. Injection of free radiolabel produced a peak with the same retention time, and trichloroacetic acid (TCA) precipitation showed there was no covalent incorporation. 
SDS-PAGE analysis revealed that approximately 95% of the HMW fraction was α-crystallin. To determine whether this was the protein interacting with dexamethasone, binding studies were performed with several concentrations of lens extract, HMW protein, and affinity-purified α-crystallin. 
The relative amounts of steroid bound by α-crystallin, HMW protein, and lens extract were the same at all protein concentrations tested. At 25 mg/ml protein, the amounts bound were approximately 700, 1490, and 1580 femtomoles/mg protein for lens extract, HMW, and α-crystallin, respectively. When the lens extract and HMW were expressed relative to their α-crystallin content (45% and 95%, respectively), bound dexamethasone corresponded to 1580 and 1560 femtomoles/mg, respectively, essentially identical with that of the pure protein. These data confirm that α-crystallin was the major soluble protein interacting with the steroids. Binding studies with the twoα -crystallin subunits, αA and αB, indicated these had similar dexamethasone-binding capacities (data not shown). 
To examine the strength of the interaction, the effects of variations in dexamethasone concentration was examined. The data in Figure 2 reveal that the association of dexamethasone with α-crystallin increased linearly over a 63-fold concentration range with no indications of saturation being reached. The backward extrapolation of the line from high concentrations passes through zero, indicating that there was no specific binding. Similar linear binding was previously observed with unfractionated lens extract. 7 The Scatchard plot of the data (Fig. 3) shows a near-horizontal line that is indicative of nonspecific partitioning. 
Corticosteroid-Binding Protein Sequence Comparison
The bovine α-crystallin polypeptide sequences were compared with those of several corticosteroid-binding proteins (CBPs). Little homology was observed, except in the yeast CBP. 23 As shown in Figure 4 , this contained three widely separated sequence segments of 5, 7, and 5 residues that exhibited a high degree of homology with sequences inα -crystallin. Of the 17 residues, 11 were identical, and a further three had amino acids with very similar properties. Each of the three sequences appeared to be highly conserved in α-crystallins and in the closely related small heat shock protein (shsp), hsp20. Unfortunately, no information is available on the significance of these to steroid binding or on the three-dimensional positioning of these areas in CBP. 
Discussion
The lens contains soluble proteins capable of associating with the glucocorticoid dexamethasone. This association is unlike that observed for glucocorticoid receptors, being heat stable, nonsaturable, and of low affinity. The data presented in this communication indicate that the majority of the dexamethasone was associated with α-crystallin. Only a very small amount was found in the β-crystallin fraction. This can be attributed to covalent modification of these proteins, which has previously been reported. 14 15  
The noncovalent interaction of dexamethasone with pure α-crystallin is best described as nonspecific partitioning. The Scatchard plot was horizontal for a wide range of concentrations, and there were no indications of a saturable component. Similar observations have previously been made with unfractionated lens extract. 7 Partitioning of small molecules into proteins is not a commonly observed phenomenon, but it takes place readily with lipid micelles. 24 α-Crystallin has micelle-like properties 25 and exhibits similar partitioning with molecules such as 1-anilinonaphthalene-8-sulfonic acid (ANS) 26 and acrylamide. 27  
The absence of a high-affinity binding site precludes any direct role for α-crystallin in steroid-mediated regulation of lens function. However, because of its ability to partition, α-crystallin could accumulate steroids that may affect steroid sensitive molecules, such as the lens membrane steroid-binding protein described by Cenedella et al. 12 Given the large amount of α-crystallin in the lens, approximately 50 mg in a human lens, such a pool could be substantial. 
The partitioning may also bring the steroid into proximity with reactive groups in the protein, principally lysine, effectively increasing the local concentration, thereby promoting adduct formation. This could explain the higher propensity of α-crystallin to form covalent steroid adducts, compared with the other crystallins. 15 It has been suggested that adduct formation produces conformational alterations that lead to cataract. However, recent work from this and other laboratories has brought into question the role of such adducts in steroid cataract. 14 15  
The ability of α-crystallin to accommodate large amounts of small molecules, with no apparent effect on its size, 27 suggests that there may be cavities within the molecule. The presence of cavities has long been suspected because of the large discrepancy between the apparent hydrodynamic volume of the protein and that expected from its molecular mass. 28 29 Recently, Haley et al., 30 using cryoelectron microscopy, observed an internal cavity in αB-crystallin aggregates, and Kim et al. 31 found a cavity in the closely related shsp Methanococcus jannischii. The cavities may participate in the nonspecific steroid partitioning observed in the present study, as well as in the partitioning of ANS 26 and acrylamide 27 and in the association with membrane lipids 32 and denatured proteins. 33  
Sequence homology was found between α-crystallin and CBP, albeit limited to three short segments. It is possible that these segments are involved in the interaction with steroids. The three-dimensional structures of α-crystallin and the CBP are unknown, but information from the closely related shsp M. jannischii’s crystal structure 31 and the polypeptide folding pattern of theα -crystallin domain 34 allow some speculation on the sites involved in the partitioning. 
The three sequences are all located in the conserved α-crystallin domain of the shsp family and correspond to the structural elements named C1, C3, and B3 by Koteiche and Mchaourab. 34 Each participates in the extensive β-pleated sheet network comprising seven strands arranged in two sheets. The C1 and B3 strands are antiparallel and lie alongside each other, generating a hydrophobic surface comprising LeuValVal from β4 and AlaLeu from β8. We suggest this surface forms part of a hydrophobic pocket into which the steroids can partition. Support for this suggestion is provided by the observation that the C1 sequence is within one of the hydrophobic sites (αA79–88) involved in the binding of ANS and in the chaperone activity. 35 The configuration of the remaining segment is unclear, because it occurs in the region where the M. jannischii shsp has nine residues more than theα -crystallins. Three-dimensional structures for both α-crystallin and CBP are required to evaluate these possibilities. 
Much remains to be learned about α-crystallin and its contribution to lens structure and function. Detailed investigations of its unique characteristics, such as the partitioning of small molecules, will provide a better insight into the role of the protein in the lens. 
 
Figure 1.
 
Separation of soluble bovine lens extract preincubated with radiolabeled dexamethasone. Bovine lens extract () was incubated with 5.2 × 10 8 M [3H] dexamethasone (•) for 3 hours. Separation was performed using GPC and fractions measured for radioactivity.
Figure 1.
 
Separation of soluble bovine lens extract preincubated with radiolabeled dexamethasone. Bovine lens extract () was incubated with 5.2 × 10 8 M [3H] dexamethasone (•) for 3 hours. Separation was performed using GPC and fractions measured for radioactivity.
Figure 2.
 
Concentration dependence of dexamethasone binding. α-Crystallin was incubated with varying concentrations (5–315 nM) of dexamethasone for 3 hours. Bound steroid was separated from free hormone using a gel chromatography column.
Figure 2.
 
Concentration dependence of dexamethasone binding. α-Crystallin was incubated with varying concentrations (5–315 nM) of dexamethasone for 3 hours. Bound steroid was separated from free hormone using a gel chromatography column.
Figure 3.
 
A Scatchard analysis of dexamethasone binding by bovine lensα -crystallin. The binding data from Figure 2 were used to construct this plot.
Figure 3.
 
A Scatchard analysis of dexamethasone binding by bovine lensα -crystallin. The binding data from Figure 2 were used to construct this plot.
Figure 4.
 
Sequence comparison between bovine αA- and αB-crystallin, human hsp20, and yeast CBP. Shown are three individual sequence motifs in which there appeared a significant degree of homology. Identical residues have been highlighted.
Figure 4.
 
Sequence comparison between bovine αA- and αB-crystallin, human hsp20, and yeast CBP. Shown are three individual sequence motifs in which there appeared a significant degree of homology. Identical residues have been highlighted.
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Figure 1.
 
Separation of soluble bovine lens extract preincubated with radiolabeled dexamethasone. Bovine lens extract () was incubated with 5.2 × 10 8 M [3H] dexamethasone (•) for 3 hours. Separation was performed using GPC and fractions measured for radioactivity.
Figure 1.
 
Separation of soluble bovine lens extract preincubated with radiolabeled dexamethasone. Bovine lens extract () was incubated with 5.2 × 10 8 M [3H] dexamethasone (•) for 3 hours. Separation was performed using GPC and fractions measured for radioactivity.
Figure 2.
 
Concentration dependence of dexamethasone binding. α-Crystallin was incubated with varying concentrations (5–315 nM) of dexamethasone for 3 hours. Bound steroid was separated from free hormone using a gel chromatography column.
Figure 2.
 
Concentration dependence of dexamethasone binding. α-Crystallin was incubated with varying concentrations (5–315 nM) of dexamethasone for 3 hours. Bound steroid was separated from free hormone using a gel chromatography column.
Figure 3.
 
A Scatchard analysis of dexamethasone binding by bovine lensα -crystallin. The binding data from Figure 2 were used to construct this plot.
Figure 3.
 
A Scatchard analysis of dexamethasone binding by bovine lensα -crystallin. The binding data from Figure 2 were used to construct this plot.
Figure 4.
 
Sequence comparison between bovine αA- and αB-crystallin, human hsp20, and yeast CBP. Shown are three individual sequence motifs in which there appeared a significant degree of homology. Identical residues have been highlighted.
Figure 4.
 
Sequence comparison between bovine αA- and αB-crystallin, human hsp20, and yeast CBP. Shown are three individual sequence motifs in which there appeared a significant degree of homology. Identical residues have been highlighted.
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