Molecular Vision 2001; 7:178-183 <http://www.molvis.org/molvis/v7/a25/> Received 14 June 2001 | Accepted 21 July 2001 | Published 26 July 2001

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Spectroscopic analysis of lens recombinant bB2- and gC-crystallin

Ling Fu, Jack J.-N. Liang
 
 

Center for Ophthalmic Research, Brigham and Women's Hospital, Department of Ophthalmology, Harvard Medical School, Boston, MA

Correspondence to: Jack J.-N. Liang, Center for Ophthalmic Research, Brigham and Women's Hospital, 221 Longwood Avenue, Boston, MA, 02115; Phone: (617) 278-0559; FAX: (617) 278-0556; email: jliang@rics.bwh.harvard.edu

Abstract

Purpose: To compare the spectroscopic and unfolding properties of human lens bB2- and gC-crystallin with those of aA-crystallin.

Methods: Human lens bB2- and gC-crystallin were cloned and measured spectroscopically. The unfolding curves in response to guanidine HCl (GdnHCl) and heat were also obtained by measuring Trp fluorescence emission intensity or emission maximum wavelength with increasing perturbation.

Results: Very similar spectroscopic and unfolding properties were seen with bB2- and gC-crystallin, but both demonstrated great differences compared with aA-crystallin. Unlike aA-crystallin, bB2- and gC-crystallin showed very little binding to Bis-ANS (4,4'-dianilino-1,1'-binaphthalene-5,5'-disulfonic acid), a hydrophobic fluorescence probe. Both bB2- and gC-crystallin were more resistant than aA-crystallin to GdnHCl-induced unfolding, but aA-crystallin was more resistant than bB2- and gC-crystallin to heat induced unfolding.

Conclusions: It was observed that bB2- and gC-crystallin showed more similar spectroscopic and unfolding properties with each other than each of them showed with aA-crystallin.

Introduction

The majority of human lens proteins consist of three major crystallins, designated as a-, b-, and g-crystallin. Each can be further fractionated into many components, such as aA and aB in a-crystallin, bA1-bA4, and bB1-bB3 in b-crystallin, and gA-gE in g-crystallin [1]. The major components in each class are aA-, bB2-, and gC-crystallin, respectively [2]. Much attention has recently been paid to a-crystallin, while studies on b- and g-crystallin are more limited. This focus reflects the finding that a-crystallin is a small heat-shock protein and can function as a chaperone molecule [3,4]. On the other hand, three-dimensional structures have been determined for many b- and g-crystallins [5-8] but not for a-crystallin. In the human lens, these crystallins become progressively less soluble with age and cataract formation, as evidenced by an increase in high-molecular-weight aggregation and insolubilization [1]. It has been suggested that a-crystallin is most susceptible to aggregation and insolubilization, but the factors contributing to this susceptibility are not known [9-11]. A comparative study of the three major crystallins may provide a clue. For this purpose, we cloned these three crystallins, which are unmodified and are in the native conformation.

Methods

Cloning bB2- and gC-crystallin

The preparation of the recombinant aA- and aB-crystallin has been described elsewhere [9], and we followed this method for preparing bB2- and gC-crystallin. Human bB2-crystallin cDNA in the plasmid pGEM and gC-crystallin cDNA in the plasmid pDIRECT were kindly provided by Dr. Paul Russell (NEI) [12] and Dr. Mark Petrash (Washington University, St. Louis, MO) [13], respectively. We performed PCR with high-fidelity pfu DNA polymerase to incorporate the NdeI site into the cDNA seuence at the ATG starting codon and to incorporate the HindIII site at the 3' end. These two sites were generated for the purpose of incorporating the gene into the expression vector pAED4. The forward PCR primer for bB2-crystallin is 5'-GACAGTCCCATATGGCCTCAGATCACC-3', and the reverse primer is 5'-CTGGGAAGGAAGCTTGGTGGGGA-3'. The forward PCR primer for gC-crystallin is 5'-CGTGTCAACCCACATATGGGGAAGATC-3', and the reverse primer is 5'-TTGGTAGTGTTAAGCTTTTTTAATACAAATCCA-3'. The restriction-site sequences in the above primers are in red. The primers were custom synthesized by Invitrogen Life Technologies (Baltimore, MD).

Amplification of target sequences by PCR was carried out with the Stratagene pfu DNA polymerase kit (Stratagene, La Jolla, CA). Typically, 50 ml of PCR mix (5 ml of 10X pfu buffer, 200 mM of each dNTP, 125 ng of each primer, 10 ng of DNA template, 2.5 U of pfu DNA polymerase) was used. The PCR product and the pAED4 were double digested with NdeI and HindIII. The digested gene and vector were then ligated, and the expression constructs pAED4-bB2 and pAED4-gC were obtained. The nucleotide sequences of bB2- and gC-crystallin cDNA in the constructs were determined by Sanger sequencing in an ABI automatic sequencing system (Perkin-Elmer Applied Biosystems Inc., Foster City, CA).

For overexpression, E. coli BL21(DE3) was transformed with the expression constructs pAED4-bB2 and pAED4-gC, respectively. The details for gene expression and protein purification have been described previously [9]. The proteins were identified and confirmed by Western blotting. The purity of the proteins was checked with SDS-PAGE, and the size was determined by FPLC gel filtration.

SDS-PAGE was performed in a slab gel (15% acrylamide) under reducing conditions according to the method of Laemmli [14]. Protein concentrations were determined by measuring the absorbance of aromatic amino acids at 280 nm (for bB2-crystallin, A^0.1%=1.74; for gC-crystallin, A^0.1%=2.14) [15].

Spectroscopic Measurements

Absorption spectra were obtained with a Perkin-Elmer spectrophotometer (model lambda 11, Perkin-Elmer, Norwalk, CT). Circular dichroism (CD) spectra were obtained with an Aviv Circular Dichroism Spectrometer (model 60 DS, Aviv, Lakewood, NJ). Five scans were recorded, averaged, and expressed in molar ellipticity ([q]) with units defined as deg-cm²-dmol^-1, using a polynomial-fitting program.

Fluorescence was measured with a Shimadzu spectrofluorometer (model RF-5301PC, Shimadzu, Columbia, MD). Tryptophan (Trp) emission was scanned with an excitation wavelength at 295 nm. The extrinsic probe, Bis-ANS (4,4'-dianilino-1,1'-binaphthalene-5,5'-disulfonic acid; h = 23 x 10³ cm^-1M^-1 at 395 nm, Molecular Probes, Junction City, OR) was used to determine the hydrophobicity [16]. For studies of unfolding, Trp emission intensities at 320 nm were obtained for samples with increasing guanidine HCL (GdnHCl) concentrations. For temperature-dependent changes, both Trp emission intensity and maximal wavelength were obtained. Temperature was controlled with a Lauda RC-6 water bath (Brinkmann Instruments, Westbury, NY).

Results

Recombinant bB2- and gC-crystallin

SDS-PAGE shows a 24- to 26-kDa band for bB2-crystallin and a 21-kDa band for gC-crystallin (Figure 1). FPLC gel filtration indicated that gC-crystallin was a monomer with a molecular size of 21 kDa and that bB2-crystallin was a dimer with a molecular size of 48 kDa (Figure 2).

Absorption and CD

Absorption spectra for the bB2- and gC-crystallin are very similar (Figure 3). Both displayed a prominent absorption shoulder at 290 nm, which is a characteristic of Trp absorption [17].

Figure 4 shows far- and near-UV CD spectra. The far-UV CD displayed a trough at 215 to 218 nm, a characteristic of b-pleated sheet conformation. The content of secondary structures (a-helix, b-sheet, b-turn, and random coil), as calculated by a SELCOM program [18], were 12, 27, 23, and 38, respectively, for bB2-crystallin and 15, 37, 20, and 28, respectively, for gC-crystallin. The a-helical contents are somewhat higher than those reported previously [17,19], perhaps due to differences in protein preparations or in methods of determination.

The near-UV CD spectra are quite different for bB2- and gC-crystallin, most likely reflecting the difference in their content of aromatic amino acid residues; bB2-crystallin has 5 Trp, 9 Tyr, and 8 Phe and gC-crystallin has 4 Trp, 14 Tyr, and 3 Phe. There may be some difference in tertiary structure, as evidenced from Trp fluorescence (see below). Both far- and near-UV CD features for bB2- and gC-crystallin are very similar to those reported previously [13,20].

Fluorescence

Trp fluorescence displayed an emission maximum at 332 nm for bB2-crystallin and at 329 nm for gC-crystallin (Figure 5A), indicating that Trp residues are buried relative to those in recombinant aA-crystallin (l_em = 336-337 nm) [9,17,21]. The results of the extrinsic probe Bis-ANS showed that the intensity for aA-crystallin is almost five-fold greater than that for bB2- or gC-crystallin (Figure 5B). The findings are consistent with the notion that a-crystallin is much more hydrophobic than b- and g-crystallin [22,23].

Unfolding study

Samples of bB2- and gC-crystallin with different concentrations of GdnHCl were left overnight at room temperature to reach equilibrium. Trp fluorescence at 320 nm was measured and shown in Figure 6. The data for aA-crystallin were included for comparison [24]. gC-Crystallin was more resistant than bB2-crystallin to GdnHCl-unfolding, which in turn was more resistant than aA-crystallin to GdnHCl-unfolding. For both bB2- and aA-crystallin, the unfolding curve showed a two-phase transition, whereas for gC-crystallin, the transition curve was monophasic and sigmoid.

Temperature-dependent changes of Trp emission intensities at maximum wavelength were plotted (Figure 7). The rate of intensity change was greater for bB2- and gC-crystallin than for aA-crystallin. gC-Crystallin precipitated at 60 °C, and fluorescence could not be measured. The change in emission maximum wavelength was a better demonstration of conformational change [17]. aA-Crystallin showed very little change throughout the entire temperature range (from 336 nm at 25 °C to 338 nm at 80 °C), but bB2-crystallin showed a drastic change starting at 50 °C and continued unfolding without precipitation at higher temperatures (from 332 nm at 25 °C to 348 nm at 80 °C). gC-Crystallin precipitated out once it started to unfold.

Discussion

Many previous studies indicate that bB2-crystallin is a dimer while gC-crystallin is a monomer. They have similar structures characterized by domains of Greek key motifs [5-8]. In monomer g-crystallin, two domains with paired motifs are each organized around a dyad; the connecting peptide folds back to let domains associate intramolecularly. In the bB2-crystallin, the domain structure is similar but the connecting peptide is extended to favor an intermolecular-domain interaction to form a dimer. The surface interactions in the bB2-crystallin dimer are similar to the intramolecular interactions in g-crystallin. The interdomain interactions, as well as the high structural symmetries, thus provide unusual stability to these proteins. Our unfolding study indeed shows that bB2- and gC-crystallin are more resistant than aA-crystallin to unfolding by GdnHCl.

The GdnHCl-unfolding curves for aA- and bB2-crystallin were biphasic, indicating that there were intermediates in the unfolding process, as reported previously [25,26]. A logical explanation is that during denaturation the quaternary structures were dissolved first, and then the tertiary and secondary structures were disrupted. The three-state model in the analysis of unfolding of aA- and aB-crystallin to obtain conformational stability (standard free energy) is not appropriate for oligomers in the previous report [24] and no attempt was made to analyze in the same way for dimer bB2-crystallin since the unfolding may involve dissociation intermediates. For monomer gC-crystallin, it is obvious that the unfolding is a two-state transition. Instead of comparing conformational stability with a- or b-crystallin, we plan to compare modified and mutant gC-crystallin with wild-type gC-crystallin. For example, we are currently studying the effects of CML [N^e-(carboxymethyl)lysine] formation and T5P mutation on the conformational stability. CML adduct is one of the major advanced glycation end products. The T5P mutation of gC-crystallin is associated with one of the autosomal dominant congenital cataracts, Coppock-like cataract, which has phenotype of a dust-like opacity of the fetal nucleus [27,28].

The mechanism of thermal unfolding is entirely different from that of GdnHCl unfolding. aA-Crystallin is thermally more stable than bB2- or gC-crystallin because of its greater hydrophobicity; the hydrophobic interaction increases at high temperature. Although thermal stability is a fundamentally important property, it is not physiologically relevant, especially at high temperatures, since human lenses are seldom exposed to such high temperatures. Many previous reports indicate that a-crystallin undergoes two transitions with changes in temperature; the first at around 30 °C and the second at around 60 °C [29,30]. This has led to speculation that the first transition involves changes in quaternary and tertiary structure and that the second transition involves a change in secondary structure. However, our Trp fluorescence data showed no secondary structural change or denaturation for aA-crystallin. On the other hand, both bB2- and gC-crystallin undergo denaturation, and gC-crystallin even precipitated out.

The significance of conformational stability for g-crystallin lies in its synthesis during the early stage of development and its greater abundance in the nucleus than in the cortical region. Since metabolism is relatively inactive in the nucleus, the abundant g-crystallin is constantly subjected to various assaults and its stable conformation thus provides a mechanism to sustain from such assaults. This is consistent with the observation that the nucleus of the old human lens contains little soluble a-crystallin [31].

Because of their homology and structural similarities, b- and g-crystallin are classified as a superfamily of b/g-crystallin. However, some subtle differences in their spectroscopic and unfolding properties were observed. The differences may be caused by different domain interaction, inter-domain interaction in bB2-crystallin and intra-domain interaction in gC-crystallin or by the presence of extensions or arms at the N- and C-termini of bB2-crystallin but not gC-crystallin. These extensions also may play a role in intermolecular interaction.

In conclusion, our studies indicate that the human major b- and g-crystallin, bB2-crystallin and gC-crystallin, have very similar conformational and stability properties but that they are relatively more stable to GdnHCl unfolding, but less stable to heat, than the major a-crystallin, aA-crystallin.

Acknowledgements

This work was supported by grants from the National Institutes of Health (EY05803) and Massachusetts Lions Eye Research Fund. The authors are very grateful to Drs. Mark Petrash and Paul Russell for supplying gC- and bB2-crystallin cDNAs.

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