Ординатура / Офтальмология / Английские материалы / Progress in Lens and Cataract Research_Hockwin_2002

Tryptophan Fluorescence

Tryptophan fluorescence was used to investigate the modifications of -crystallin solutions (100 g/ml) further. Solutions were excited at 295 nm, slit width 10 nm, and emission spectra of between 300 and 500 nm were recorded. In further experiments the fluorescence (excitation 320 nm, emission 340–500 nm) of solutions was recorded on a Perkin Elmer LS 50 B luminescence spectrometer. Absorbance readings of the -crystallin samples were measured at various wavelengths on a UVIKON 930 Kontron spectrophotometer.

Statistical Analysis

All results were subjected to a paired Student t test where relevant.

Results

Effect of Modification on Protection against the Thermally Induced Aggregation of L-Crystallin

The thermally induced aggregation of L-crystallin proceeded rapidly in the absence of -crystallin (fig. 1), optical density reaching a maximum of 0.88 after 46.2 min (data not shown). Unmodified -crystallin suppressed the aggregation of L-crystallin and protection was found to be statistically significant after 20 min (p 0.044). Modified -crystallin samples protected againstL-crystallin aggregation to differing extents. Protection by (25 mM fructose) and (25 mM p-21-h) was statistically significant after just 20 min (p 0.027 and 0.017, respectively) whereas protection by (10 mM p-21-h) was statistically significant after 30 min (p 0.001). In the case of (25 mM p-21-h) protection against the thermally induced aggregation of L-crystallin was not statistically significant after 40 min (p 0.188).

Modified -crystallin samples were statistically less effective in protectingL-crystallin against thermally induced aggregation. After 40 min, the optical density of the L-crystallin solution containing unmodified -crystallin was 0.187, whereas the optical densities of solutions containing the replacements of-crystallin samples, (25 mM fructose), (10 mM p-21-h) or (25 mM p-21-h), were 0.372 (p 0.01), 0.498 (p 0.042) and 0.671 (p 0.039), respectively.

Effect of Modification on Protection against the Thermally Induced Aggregation of ADH

The thermally induced aggregation of ADH proceeded rapidly in the absence of -crystallin (fig. 2), reaching a maximum of 0.886 after 26.8 min (data not shown). Unmodified -crystallin suppressed the aggregation of ADH and protection was found to be statistically significant after 10 min (p 0.01). Protection after 10 min by the modified samples (25 mM fructose) and(25 mM p-21-h) was also significant (p 0.01).

Although it appears from figure 2 that unmodified -crystallin might be less effective than the modified samples at protecting against the thermal aggregation of ADH, the difference in protection between samples was not statistically significant.

Effect of Modification on Protection against the Thermally Induced Aggregation of Glyceraldehyde 3-Phosphate Dehydrogenase and Catalase

Glyceraldehyde 3-phosphate dehydrogenase is labile at 37 °C in triethanolamine buffer and at this temperature the enzyme rapidly precipitates out of solution. This gave us the opportunity to perform an aggregation assay in the presence

of -crystallin at the physiological temperature of 37 °C. Previously Lee et al. [32] demonstrated that stable complex formation between GAP-DH and HSP 18.1, the small heat shock protein from pea, could occur at temperatures as low as 34 °C [32]. As can be seen from figure 3, glyceraldehyde 3-phosphate dehydrogenase rapidly precipitates out of solution in triethanolamine buffer at 37 °C. -Crystallin at a stoichiometric ratio of :GAP-DH 1:1 w:w completely suppressed this aggregation at 37 °C (data not shown). At a stoichiometry of 1:2 w:w normal-crystallin conferred significant but suboptimal protection (fig. 3).

Unmodified -crystallin and -crystallin modified by 25 mM fructose 6-phosphate, 10 mM p-21-h or 25 mM p-21-h all significantly protected GAPDH against precipitation after 60 min (p 0.021, 0.030, 0.044 and 0.027, respectively). There was no statistically significant difference in the protection conferred by the various -crystallin samples against GAP-DH precipitation throughout the time course of the incubation (fig. 3).

In the catalase thermal aggregation assay at 55 °C, catalase was found to precipitate rapidly out of solution reaching a maximum turbidity after 38.4 min (data not shown). Protection by all -crystallin preparations against this aggregation was statistically significant after 20 min for the unmodified -crystallin solutions and for the -crystallin solutions modified by 25 mM fructose 6-phosphate, 10 mM p-21-h and 25 mM p-21-h solutions (p 0.18, 0.18, 0.024 and 0.040, respectively) (fig. 4). There was no statistically significant difference in protection against catalase aggregation between the -crystallin solutions at the 10-min time intervals examined.

Tryptophan Fluorescence

Analysis of the optical density readings of the various -crystallin solutions demonstrated that the modified -crystallin solutions had higher absorption

spectra in the range of 260–340 nm. Compared to the unmodified -crystallin solution, solutions that had been modified by 25 mM fructose, 10 mM p-21-h or 25 mM p-21-h had increased absorbance readings of 27, 34 and 108% at 280 nm and of 15, 30 and 70% at 329 nm. Fluorescence spectroscopy shown in figure 5 demonstrated that the tryptophan fluorescence of modified -crystallin samples was significantly decreased compared to the unmodified sample. This indicates that a conformational change has occurred within the environment of the tryptophan residues of -crystallin. The effect was particularly prominent in-crystallin samples modified by the glucocorticoid p-21-h. This is not due solely to inner filtering because upon excitation at 280 nm, the fluorescence of the unmodified -crystallin solution at the emission wavelength of 329 nm was 45, 475 and 726% greater in magnitude than that of the -crystallin solutions

that had been modified by 25 mM fructose, 10 mM p-21-h and 25 mM p-21-h, respectively. These intensity differences between the unmodified -crystallin solution and the modified -crystallin solutions are much greater than the differences in absorbance of the unmodified -crystallin solution compared to the modified solutions mentioned above. The max of the unmodified -crystallin solution and all the modified -crystallins was between 329 and 329.5 nm.

Discussion

Generally the role of small heat shock proteins in vivo is thought to be primarily concerned with the prevention of aggregation of unfolding or misfolded proteins. Classically, this can be observed in the laboratory in thermal aggregation assays. We have utilized such assays to study the effect of posttranslational modifications on chaperone activity. In previous experiments -crystallin’s chaperone function has appeared to be relatively resistant to disruption by chemical modification. Whereas enzyme function can be completely compromised within several days or hours of incubation with low concentrations of sugars [6, 14, 15, 33, 34], cyanate [35, 36] or steroids [16, 36], -crystallin’s chaperone function is totally resistant to several modifications, including glycation by 100 mM fructose or 100 mM glucose 6-phosphate and carbamylation by 25 mM sodium cyanate over incubation periods of up to 10 days [27]. However van Boekel et al. [27] did see chaperone function compromised upon incubation with 100 mM ribose which resulted in 21% of the -crystallin subunits being cross-linked. These results indicated that late glycation products, capable of cross-linking proteins, could decrease the chaperone activity of -crystallin. Glycation of -crystallin by erythrose was shown to compromise chaperone function after just 1 day of incubation in one study [20]. However, the authors of this study provided no statistical evidence for the small difference in chaperone protection observed between unmodified and glycated -crystallin samples. They also did not indicate whether free sugar had been dialysed out of the glycated -crystallin solution after the incubation period was completed and prior to assays being performed. In our laboratory we have observed that the presence of free sugar in solutions can lead to increased enzyme inactivation at the elevated temperatures found in the thermal aggregation assays [Hook and Harding, unpubl. data].

In the study communicated in this paper, we found that -crystallin chaperone function was significantly compromised upon incubation with fructose 6-phosphate or p-21-h as based on the L-crystallin aggregation assay. However the ability of modified -crystallin solutions to protect against enzyme aggregation in any of three thermal aggregation assays was not decreased.

The stoichiometry of protection by -crystallin against thermally induced aggregation of proteins may be more efficient for protection of L-crystallin than for enzymes [4]. However, catalase is protected by -crystallin as efficiently as L-crystallin in thermal aggregation assays (in terms of w:w ratio), yet chaperone function as judged in this assay was not compromised.

Why catalase was much more efficiently protected may relate to the relative temperature at which the assay was performed. For example, the GAP-DH, ADH, L-crystallin and catalase assays were performed at 37, 48, 55 and 55 °C, respectively. At higher temperatures more hydrophobic sites would be expected to be available for interaction with denatured molecules. Indeed the chaperone activity of -crystallin has been shown to increase with temperature [37, 38]. Our GAP-DH assay protocol at 37 °C will allow further investigation of the structure/function relationship of -crystallin to be investigated under physiological conditions.

Tryptophan fluorescence spectroscopy provided evidence that -crystallin samples had been modified by both fructose 6-phosphate and p-21-h after 25 and 12 days, respectively. All modified -crystallin solutions were markedly quenched compared to the unmodified -crystallin solution and this quenching, at least in the case of (p-21-h) solutions, was not due to an internal filtering effect. These results may indicate that a conformational change has occurred within the environment of a tryptophan residue of the modified -crystallin samples which could implicate that a modification has taken place close to a tryptophan residue.

This paper has demonstrated that protection against L-crystallin aggregation by -crystallin can be compromised upon prolonged exposure to p-21-h or fructose 6-phosphate. This may have implications for the ability of -crystallin to protect against aggregation when steroid or sugar levels are raised within the lens as a result of a glucocorticoid therapy treatment or upon the onset of diabetes. Loss of this protective effect may lead to cataract.

Acknowledgment

We are grateful to the Wellcome Trust for financial support.

References

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Prof. J.J. Harding, Nuffield Laboratory of Ophthalmology, University of Oxford, Walton Street, Oxford OX2 6AW (UK)

Tel. 44 1865 248996, Fax 44 1865 794508, E-Mail john.harding@eye.ox.ac.uk

physiological and pharmacological activities in the living system. We assume that the steroidinduced cataract is one of the adverse effects caused by synergic biological activities of glucocorticoids.

Copyright © 2002 S. Karger AG, Basel

Introduction

Black et al. [1] reported that cataract was observed as one of the adverse effects of glucocorticoid therapy in humans. However, attempts in animals have been unsuccessful in producing cataract with glucocorticoids. In 1983, we demonstrated that glucocorticoids frequently caused a decline of glutathione (GSH) and a loss of transparency in the lens of developing chick embryos [2]. During our experiments, we described that the cataract formation can be observed during the treatment with steroids possessing biological activities of glucocorticoids and probably produced by lipid peroxide (LPO, TBA-reacting substances) synthesized in the liver after glucocorticoid administration [3–7]. Recently, Shui et al. [8, 9] reported that posterior cataract can be induced in rats with a combination treatment of prednisolone and X-ray irradiation.

On the other hand, there are several experiments using the cultured lens and the lens proteins treated with steroids. Manabe et al. [10] and Bucala et al. [11] suggested that the formation of Schiff bases between the steroid C-20 carbonyl group and ε-amino groups of crystallin lysine residues, followed by a Heyns rearrangement involving the adjacent C-21 hydroxyl residue, was involved in steroid-induced cataract formation. However, this conception is not always generally accepted at the present time because lens epithelial cells of bovines, rats and young humans contained a 28-kD protein that could bind progesterone (no hydroxyl residue at C-21 position) and perhaps other steroid hormones including glucocorticoids with a high affinity [12]. Moreover, with detailed experiments using lens protein -crystallin and lens culture studies, Dickerson et al. [13] indicated that nonglucocorticoids such as progesterone could bind lens proteins as well as or better than dexamethasone, and that only glucocorticoids, not other steroids, lowered the lens GSH content as demonstrated in many other forms of cataract. These experiments attempt to elucidate the mechanism of cataract formation based on direct effects of the steroid on the lens. However, it should be noted that glucocorticoids cause various metabolic changes in many tissues. Therefore, it is quite likely that some adverse effects after glucocorticoid treatment are caused by changes in blood components rather than the direct effect of the glucocorticoid on the lens.

In the present paper, in order to investigate the mechanism of cataract formation, the chick embryo lenses, which became cataractous in developing