|Molecular Vision 2000;
Received 26 October 1999 | Accepted 24 February 2000 | Published 2 March 2000
Heat-induced conformational change of human lens recombinant aA- and aB-crystallins
Jack J-N Liang, Tian-Xiao
Sun, Nila J. Akhtar
Center for Ophthalmic Research, Brigham and Women's Hospital, Harvard Medical School, Boston, MA
Correspondence to: Jack J-N Liang, Center for Ophthalmic Research, 221
Longwood Avenue, Boston, MA, 02115; Phone: (617) 278-0559; FAX: (617)
Dr. Sun is now with the Renal Unit of the Massachusetts General Hospital/East, Charlestown, MA, 02129
Purpose: To determine which component of lens a-crystallin is responsible for heat-induced transition, conformational change and high molecular weight (HMW) aggregation.
Methods: Recombinant aA- and aB-crystallins were used. Temperature dependent changes were probed by Trp fluorescence and circular dichroism (CD) measurements. HMW aggregates were induced by heating at 62 °C for 1-2 h and then cooling to room temperature. The nature of HMW aggregation was studied with fluorescent probes, 4,4'-dianilino-1,1'-binaphthalene-5,5'-disulfonic acid (bis-ANS) and thioflavin T (ThT).
Results: CD and Trp fluorescence revealed that aB-crystallin was more susceptible than aA-crystallin to heat-induced conformational change and aggregation. At temperatures greater than 70 °C, aB-crystallin precipitated but aA-crystallin remained soluble. Both bis-ANS and ThT probes displayed increased fluorescence intensity with HMW aggregation, but the increase for bis-ANS was greater with aB-crystallin than with aA-crystallin, while the reverse was true for ThT.
Conclusions: These results indicate that aB-crystallin is more susceptible than aA-crystallin to heat-induced conformational change and aggregation and are consistent with the notion that aA- and aB-crystallins have different biochemical and biophysical properties in spite of their high degree of homology.
a-Crystallin is a major crystallin of mammalian lenses and is believed to play a prominent role in the maintenance of lens transparency. It consists of two subunits, aA and aB, which form an oligomer with a molecular weight of approximately 800 kDa . Given the protein's unique properties, interest in a-crystallin has greatly intensified recently. a-Crystallin has been reported to possess a chaperone-like property and a high degree of homology to the small heat-shock protein [2-4]. Moreover, aB-crystallin is expressed in many non-lenticular tissues, and it's expression increases in some neurological diseases [5-7]. The greater non-lenticular expression and the greater chaperone-like activity [8-10] of aB-crystallin than of aA-crystallin suggest the significance of differential function or conformation between aA- and aB-crystallins.
Studies indicate that native a-crystallin is thermally stable [11,12]. It undergoes a minor thermal transition at 40 °C and a major thermal transition at 60 °C, but no denaturation is observed even at 90 °C . The transition at 60 °C was reported subsequently to be partial unfolding, and incubation at high temperatures results in irreversible high-molecular-weight (HMW) aggregates . Recently, we studied cloned human lens aA- and aB-crystallins and found that aB-crystallin was more susceptible to heat-induced aggregation than aA-crystallin ; the implication is that the aA- and aB-crystallins respond differently to heat. We also have noted other differences between aA- and aB-crystallins, including differences in hydrophobicity, chaperone-like activity, and rate of subunit exchange [8,15]. In this work, we further study heat-induced conformational change and HMW formation with circular dichroism (CD) and fluorescence measurements. The results indicate a greater change in near-UV CD and Trp fluorescence for aB- than for aA-crystallin. The greater susceptibility to conformational change may be related to its greater chaperone-like activity and heat- and stress-inducible properties.
Human lens recombinant aA- and aB-crystallins were prepared from a cDNA library of epithelial cells . These proteins are present in a native state with a molecular weight of 600-800 kDa. gB-Crystallin was prepared from calf lens as described previously [16,17]. Protein concentrations were determined by absorption based on aromatic amino acid composition .
Fluorescence probes bis-ANS and ThT were purchased from Molecular Probes (Johnson City, OR) and Sigma (St. Louis, MO), respectively.
HMW aggregates were formed by heating of either aA- or aB-crystallin at 62 °C for 2 h. FPLC gel filtration indicated the formation of HMW aggregates (data not shown), as shown in our earlier studies (12,14).
CD spectra were measured with an Aviv Circular Dichroism Spectrometer (model 60 DS). Spectra of five scans were averaged and smoothed by a polynomial-fitting program .
Fluorescence was measured with a RF-5301PC Shimadzu spectrofluorometer. Trp emission spectra were scanned with an excitation wavelength of 290-295 nm.
Bis-ANS fluorescence emission spectra were scanned between 460 and 560 nm with an excitation wavelength of 395 nm. Aliquots of 20 ml of bis-ANS (100 mM stock solution) were added to a-crystallin solution (0.1 mg/ml in 50 mM phosphate buffer, pH 7.5) until saturation was reached. The samples were incubated for 10 min at room temperature before fluorescence measurement. ThT fluorescence emission spectra were obtained in a manner similar to that used for bis-ANS, but with an excitation wavelength of 450 nm and scanning between 470 and 520 nm.
The desired temperatures for CD and fluorescence measurement were maintained with a Lauda RC6 water-bath (Brinkmann Instruments, Westbury, NY). The equilibrium time at each temperature was 15 min.
The representative far- and near-UV CD spectra are shown in Figure 1 and Figure 2, respectively. An increased far-UV CD intensity (toward a more negative signal) and a decreased near-UV CD intensity were observed at high temperatures. While the change in far-UV CD is similar for the aA- and aB-crystallins, the decrease of near-UV CD was dramatic for aB-crystallin (Figure 3). At temperatures greater than 70 °C, aB-crystallin was precipitated, but the solution of aA-crystallin remained clear.
With increasing temperature, Trp emission intensity decreased and the emission maximum shifted to longer wavelengths (Figure 4). Emission intensity decreased at the emission maxima (Figure 5); the decrease was greater for aB-crystallin than for aA-crystallin. These changes are related to partial unfolding as the Trp residues became more exposed.
Bis-ANS and ThT fluorescence
Figure 6 shows bis-ANS and ThT fluorescence spectra for aA- and aB-crystallins as well as gB-crystallin. With the hydrophobic probe bis-ANS , aB-crystallin displayed a greater intensity than aA-crystallin, and gB-crystallin gave hardly any fluorescence. With ThT, the intensity for aA- was greater than for aB-crystallin. ThT is a probe specific to aggregation by proteins rich in b-sheet conformation, such as b-amyloid fibril [20,21]. gB-Crystallin, though rich in b-sheet conformation, did not display ThT fluorescence because of it's monomer composition.
Bis-ANS and ThT fluorescence spectra of HMW aA- and aB-crystallin aggregates are shown in Figure 7 and Figure 8, respectively. Both intensities increased with HMW formation. aB-crystallin displayed a greater increase in bis-ANS intensity than aA-crystallin, while the reverse was true for ThT fluorescence.
We have reported that aB-crystallin is more susceptible to heat-induced aggregation than aA-crystallin and that addition of aA- to aB-crystallin reduces the extent of aB-crystallin aggregation . aB-crystallin's greater tendency toward aggregation at high temperatures is apparently caused by its greater susceptibility to conformational change; i.e., aB-crystallin becomes more unfolded than aA-crystallin. The initial partial unfolding of proteins is usually followed by aggregation. The fact that aB-crystallin is a small heat-shock protein and is heat and stress inducible may be related to its greater susceptibility than aA-crystallin to partial unfolding. This partial unfolding enhances chaperone-like activity but also promotes hydrophobic interaction among protein molecules and may lead to HMW aggregation . In the heterogeneous a-crystallin, this effect is counterbalanced by a rapid subunit exchange between aA- and aB-crystallin [14,15,22]. The loss of stability of aB-crystallin has also been demonstrated in studies with aA-crystallin knockout mice, in which most aB-crystallin is present in exclusion bodies . In this regard, it would be interesting to see what form of aB-crystallin is expressed in other tissues.
Partial unfolding by heat is apparent from the results of fluorescence and near-UV CD. A decreased Trp emission intensity and a red shift of emission maximum, along with a decreased near-UV CD, indicate that proteins become partially unfolded at elevated temperatures. The greater shift in the unfolding curves by Trp fluorescence (Figure 5) as well as by near-UV CD (Figure 3) with increasing temperature for aB-crystallin than for aA-crystallin indicated that partial unfolding was greater for aB-crystallin than for aA-crystallin.
The mechanism underlying the formation of HMW aggregates of aA- and aB-crystallins may be similar to that of b-amyloid fibril formation, which involves increasing of b-sheet conformation and formation of fibril . ThT has been widely used in the detection of fibril formation of b-amyloid. The display of far less ThT fluorescence by gB-crystallin than by aA- and aB-crystallins indicates that both b-sheet conformation and aggregation are required for ThT fluorescence. Further aggregation enhances ThT fluorescence.
The observation of a-crystallin as the major crystallin in the water insoluble fraction of human aged and cataractous lenses suggests that a-crystallin is more susceptible to conformational change and aggregation than the other crystallins [25,26]. This possibility is further supported by the absence of soluble a-crystallin in the nucleus of the aged human lens . These observations must be related to the differences in conformational stability among the three major crystallins. A determination of the standard free energy DGH2O, the standard free energy change for the unfolding transition in the presence and absence of denaturant, provides a quantitative evaluation. We have determined the DGH2O value for gF-crystallin  as well as for aA- and aB-crystallins . gF-Crystallin is more stable (DGH2O = 8.9 kcal/mol) than either aA- or aB-crystallin (DGH2O = 6.4 and 5.0 kcal/mol, respectively). The difference in stability between aA- and aB-crystallins may be explained in the difference of their DGH2O values, although heat unfolding may not always be the same as chemical unfolding.
Another unresolved issue is whether the conformationally less stable aB-crystallin is preferentially insolubilized in the lens. A report by Truscott et al.  suggested that this could be the case; they reported that the insoluble, cross-linked, and colored cataract protein contained more aB-crystallin than aA-crystallin, an observation indicating that aB-crystallin was more susceptible to modification than aA-crystallin.
In conclusion, we have demonstrated that aB-crystallin is more susceptible than aA-crystallin to heat-induced conformational change and HMW aggregation. The significance of this observation may be related to the enhanced expression and chaperone-like activity of aB-crystallin under certain stresses. Earlier studies indicate that partial unfolding and HMW aggregation of a-crystallin increase hydrophobicity and chaperone-like activity [12,30,31]. Under stress, such as heat, the partially unfolded state and great chaperone-like activity of the increased aB-crystallin may confer thermotolerance to the cells.
This work was supported by a grant from the National Institutes of Health (EY05803).
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