|Molecular Vision 2001;
Received 29 May 2001 | Accepted 18 July 2001 | Published 26 July 2001
Analysis of a-crystallin chaperone function using restriction enzymes and citrate synthase
Santhoshkumar,1 Krishna K.
Departments of 1Ophthalmology and 2Biochemistry, University of Missouri, Columbia, MO
Correspondence to: Krishna K. Sharma, Departments of Ophthalmology and Biochemistry, University of Missouri, EC 213, 1 Hospital Drive, Columbia, MO, 65212; Phone: (573) 882-8478; FAX: (573) 884-4100; email: Sharmak@health.missouri.edu
Purpose: To compare the abilities of aA-crystallin, aB-crystallin, and mini-aA-crystallin (a synthetic peptide chaperone representing the functional unit of aA-crystallin) to protect against heat-induced inactivation of citrate synthase (CS) and restriction enzymes, SmaI and NdeI.
Methods: Restriction enzymes, SmaI and NdeI were heated at different temperatures in the presence of various amounts of molecular chaperones and tested for their ability to cleave plasmid DNA. The aggregation of CS was measured at 43 °C while the loss in activity was monitored at 37 °C in the presence of various crystallins.
Results: Restriction enzyme activities were protected by the crystallin subunits up to 37 °C for SmaI and 43 °C for NdeI. However, the mini-aA-crystallin was unable to protect endonuclease activity. The crystallin subunits and the peptide chaperone were able to suppress thermal aggregation of CS at 43 °C, but failed to stabilize its activity at 37 °C.
Conclusions: The ability of a-crystallin subunits to stabilize denaturing proteins varies from enzyme to enzyme as evidenced by the inactivation of CS and protection of SmaI and NdeI activity in the presence of a-crystallin subunits. Additionally, our results show that there could be more than one site in aA-crystallin responsible for its chaperone-like action. By addition of crystallin subunits to restriction enzymes prior to or during storage, transport, or assay would maintain or improve their activity thereby decreasing their cost.
a-Crystallin, a major eye lens protein and a member of the small heat shock protein family, has been shown to protect proteins from aggregation under denaturing conditions [1-4]. a-Crystallin is made of two types of subunits, aA- and aB-crystallin, and both show chaperone-like activity. The chaperone action involves hydrophobic interactions with denaturing proteins , and the sequences in aA- and aB-crystallin involved in the chaperone action have been mapped [6-8]. Recently we synthesized a peptide with a sequence similar to that of the chaperone site of aA-crystallin and showed that it can prevent the aggregation of denaturing proteins . This peptide chaperone (mini-aA-crystallin), like the whole protein, was able to prevent DTT-induced aggregation of insulin and a-lactalbumin, thermal aggregation of alcohol dehydrogenase (ADH), and UV- and oxidation-induced aggregation of g-crystallin [9-11]. Although a-crystallin prevents the aggregation of several enzymes, protection of the biological activity of only a few enzymes has been reported. While a-crystallin was unable to reactivate heat-inactivated ADH and guanidine hydrochloride (GuHCl)-denatured rhodanese, it appeared to restore partial activity of GuHCl-denatured xylose reductase, glyceraldehyde-3-phosphate dehydrogenase, and heat-inactivated citrate synthase in the presence of ATP [12-16]. Recently it has been shown that a-crystallin can prevent the heat-induced inactivation of the restriction enzyme NdeI .
The stability of restriction enzymes under storage and reaction conditions is of great concern to scientists who purchase these enzymes for research [18-20]. Restriction enzymes tend to aggregate during isolation and storage or under assay conditions, resulting in the loss of activity [21,22]. Addition of certain solvents and detergents increases the stability of these enzymes [23-26]. However, they often cause relaxation in specificity. The resulting activity is termed "star activity" [27,28]. Acetylated BSA and casein have also been shown to stabilize the activity of restriction enzymes [25,29]. These proteins, however, are poor chaperoning agents and offer only a partial stabilizing effect when used in high concentrations.
In the present study, we have analyzed the ability of aA- and aB-crystallin subunits to stabilize the activity of citrate synthase (CS) and the restriction enzymes SmaI and NdeI against thermal denaturation. We have also measured the stability of these enzymes in the presence of mini-aA-crystallin in order to see whether the anti-aggregation activity domain of aA-crystallin by itself can stabilize the biological activity of the enzymes.
Mini-aA-crystallin (DFVIFLDVKHFSPEDLTVK) was obtained from Research Genetics Inc. (Huntsville, AL). The sequence corresponds to residue 70-88 of aA-crystallin with the Lys70 replaced by Asp to increase the solubility. Restriction enzymes SmaI and NdeI, and the molecular weight markers were purchased from New England Biolabs, Inc. (Beverly, MA). The enzymes were supplied by the manufacturer in buffer containing 200 mg/ml bovine serum albumin (BSA) and 50% glycerol. Human proliferating cell nuclear antigen gene cloned in plasmid pGEM-3 (American Type Culture Collection, Manassas, VA) was used as substrate for both restriction enzymes.
Isolation of aA- and aB-crystallin
a-Crystallin was isolated from bovine lens cortex by gel filtration on Sephadex G-200 and further purified by passing through a trimethylaminoethyl fractogel column . The a-crystallin thus obtained was >99% pure, as judged by SDS-PAGE. The individual crystallins (aA- and aB-crystallin) were separated by high-pressure liquid chromatography (HPLC) using a Vydac C18 preparative column with a 0-80% water and acetonitrile gradient containing 0.1% trifluoroacetic acid. The peaks corresponding to aA- and aB-crystallin were pooled and dried. The dried samples were dissolved in 50 mM phosphate buffer (pH 7.4) containing 6.0 M urea. Urea was removed by extensive dialysis against 50 mM phosphate buffer to facilitate the refolding of aA- and aB-crystallin. SDS-PAGE of these samples showed that the refolded proteins were free of other contaminants. The concentration of a-crystallin was determined using an absorption coefficient of 0.83 for 1 mg/ml protein . The concentrations of aA- and aB-crystallin were determined using amino acid analysis  and with molar extinction coefficients of 16,500 (for aA-crystallin) and 19,000 (for aB-crystallin) at 280 nm .
Restriction enzyme activity assay
A working stock (3 units/10 ml) was prepared by diluting a known amount of the enzyme with 1X reaction buffer. All assay tubes had equal amounts of BSA (33 ng) and glycerol (0.5%) since we did not remove these from the enzyme supplied by the manufacturer. To 10 ml samples of the working enzyme in micro-centrifuge tubes were added various amounts (see figure legends) of stabilizing proteins in 1 ml of buffer, and the reaction volumes were adjusted to 13 ml with 1X buffer. The tubes were incubated at various temperatures (see figure legends). After thermal inactivation, 2 ml (200 ng) plasmid DNA was added and the tubes were incubated for 90 min at 25 °C and 37 °C for SmaI and NdeI, respectively. The digested mixtures were run on agarose gels (1%), stained with ethidium bromide, and profiles were compared with respective controls.
The chaperone assay was done as described earlier, using CS (Boehringer Mannhein, GmbH, Germany) as the substrate . Briefly, 75 mg of CS in 1 ml of 40 mM HEPES-NaOH buffer (pH 7.5) was heated at 43 °C in the presence or absence of aA-, aB- and mini-aA-crystallin (25 and 50 mg). The scattering of light was measured at 360 nm for 1 h.
Thermal inactivation of CS
CS activity was determined as described by Buchner et al. . In brief, 10 mg of CS was diluted 1000-fold in TE buffer (50 mM Tris, 2 mM EDTA, pH 8.0). Ten ml of the diluted enzyme (100 ng) was aliquoted into micro-centrifuge tubes containing 930 ml of TE buffer. The tubes were incubated at 37 °C in the presence or absence of molecular chaperones (1 mg in 10 ml TE). CS activity was determined at different time intervals after the addition of 10 ml of 10 mM oxaloacetic acid, 10 ml of 10 mM 5,5'-dithio-bis(2-nitrobenzoic acid) and 30 ml of 5 mM acetyl-CoA in TE buffer. The increase in absorbance at 412 nm was measured on a Jasco spectrophotometer (V-530, Jasco Inc., Easton, MD) for 90 s.
Thermal inactivation of restriction endonucleases
SmaI and NdeI have different levels of thermostability. SmaI loses complete activity after 60 min of incubation at 37 °C, whereas NdeI is inactivated at 43 °C after 90 min (Figure 1). We tested the stabilization of SmaI and NdeI enzyme activity using various concentrations of aA-, aB- and mini-aA-crystallin. Figure 2 shows SmaI digestion of plasmid DNA in the presence or absence of crystallin subunits at 37 °C. Addition of aA- and aB-crystallin to the reaction mixture prevented the restriction enzyme from heat inactivation. At a concentration of 0.2 mg in the assay mixture, aA- and aB-crystallin completely protected 3 units of SmaI enzyme activity. A similar observation was made with NdeI (not shown).
To measure the extent of protection of NdeI and SmaI by chaperones at various temperatures, we performed thermal inactivation assays of SmaI and NdeI in the presence of a-crystallin subunits at three different temperatures. In brief, the restriction enzymes were incubated at 37 °C, 45 °C, and 55 °C for 1.5 h in the presence or absence of aA- and aB-crystallin. Following incubation, the residual activity was determined using plasmid DNA as the substrate. DNA digestion profiles of SmaI and NdeI incubated at different temperatures are shown in Figure 3 and Figure 4, respectively. Both aA- and aB-crystallin were able to protect SmaI activity at 37 °C only. In the case of NdeI, aA- and aB-crystallin provided total protection of enzyme activity at 37 °C and a significant protection at 45 °C. Marginal protection of NdeI was seen at 55 °C by aA-crystallin. Unlike aA- and aB-crystallin, mini-aA-crystallin failed to protect against the thermal inactivation of both SmaI and NdeI.
To test whether other proteins can also stabilize endonuclease activity, we measured thermal inactivation of SmaI in the presence of BSA and lysozyme. While the incubations with lysozyme showed no activity, BSA (0.5 mg) showed partial protection from inactivation (Figure 5). The concentration of BSA required to stabilize SmaI activity was higher than that of crystallin subunits.
Thermal inactivation of citrate synthase (CS)
CS aggregates and scatters light after incubation at 43 °C. Addition of crystallin subunits or mini-aA-crystallin prevents this aggregation, and the reaction mixture remains clear (Figure 6). In order to have a rational comparison with restriction enzyme experiments, we measured the thermal inactivation of CS at 37 °C in the presence or absence of chaperones. CS readily inactivates at 37 °C. Within seconds after the start of incubation, the CS starts to lose activity, and by 45 min about 90-95% of the activity is lost (Figure 7). Unlike restriction enzymes, the presence of aA- or aB-crystallin in the assay mixture had no effect on the thermal inactivation of CS.
Protection of restriction enzymes by a-crystallin has been reported earlier . However, unlike the previous study, our study compared the protective action of individual subunits of a-crystallin with two different enzymes having different degrees of thermo-tolerance. We also tested the protective action of mini-aA-crystallin, which has the anti-aggregation properties of the parent protein . The failure of mini-aA-crystallin to protect the activity of SmaI and NdeI during the study (Figure 2, Figure 3, and Figure 4) indicates that aA-crystallin has more than one chaperone site for denaturing proteins. Earlier, mellitin-binding studies suggested that residues 13-21 in aA-crystallin might also have a role in chaperone action . Truncation or mutation in the C-terminal region was also shown to affect the chaperone-like activity of aA-crystallin . Identifying the binding sites for these enzymes in crystallin subunits would help us to design a peptide that can substitute for these proteins in assay mixtures.
Each individual a-crystallin subunit (aA- and aB-crystallin) was able to protect restriction enzymes NdeI and SmaI when incubated at 37 °C for more than 24 h. However, they failed to restore activity if added to denatured enzyme (data not shown). Of the two enzymes studied, NdeI was more stable and thus greater protection was seen with crystallin subunits. SmaI, with a half-life of 15 min at 37 °C, may be more unfolded at higher temperatures. Thus, the protective action of a-crystallin also seems to depend on the stability of the enzymes at the incubated temperature. Lysozyme, assayed for its protective effects, could not maintain SmaI activity, while BSA, when used at 2.5 fold excess the crystallin subunits, showed partial activity.
We wanted to test the protective action of crystallin subunits against thermal inactivation of other enzymes. CS was used as another enzyme substrate to study the protective action of chaperones. Each crystallin subunit (aA-, aB-, and mini-aA-crystallin) suppresed the aggregation of CS at 43 °C (Figure 6). We did not monitor the thermal aggregation of restriction enzymes, as it would have been expensive. However, the denaturation of the protein is indicated by loss of activity. In order to have a rational comparison with restriction enzymes, we measured the thermal inactivation of CS in the presence and absence of crystallin subunits at 37 °C. The activity of CS, unlike that of restriction enzymes, was not stabilized by a-crystallin subunits (Figure 7). Earlier, on the basis of tryptophan emission spectra, we had reported that there is only a partial unfolding of CS during heat denaturation . After the report by Horwitz of the chaperone-like activity of a-crystallin , several studies were directed toward measuring the protective action of this protein on enzymes. Harding and co-workers have reported that a-crystallin can reactivate catalase, 6-phosphogluconate, and glyceraldehyde-3-phosphate dehydrogenase [34-37]. a-Crystallin also assists the partial reactivation of GuHCl-denatured xylose reductase  and heat-inactivated CS in the presence of ATP [15,16]. However, it failed to protect rhodanese and ADH activity [12,13]. In the present study, we did not see protection or reactivation of CS enzyme activity by crystallin subunits. It is possible that the crystallin subunits may be binding to the active site of the enzyme during chaperone action, and reactivation would require dissociation of the complex. Earlier reports [15,16] on the reactivation of CS by a-crystallin were done either in the presence of other chaperones or ATP. Recently, we have observed that, during chaperone action, a-crystallin binds to the active site in ADH [unpublished data].
We conclude that the stabilization action of a-crystallin subunits differs with respect to the target enzymes used. The inactivation of CS in the presence of a-crystallin subunits and the protection of restriction enzymes SmaI and NdeI by a-crystallin supports this view. Further, the extent of protection seems to depend on the relative stability of the enzymes at the incubation temperature as the relatively more stable NdeI was better protected by a-crystallin from thermal inactivation than the SmaI. Further studies are required to determine whether this is due to the differences in binding of the enzymes to a-crystallin.
This work was funded by National Institutes of Health grant EY 11981. We are grateful to Dr. S. K. Swamynathan for his help.
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