|Molecular Vision 2005;
Received 22 March 2005 | Accepted 25 April 2005 | Published 30 April 2005
Interaction and biophysical properties of human lens Q155* βB2-crystallin mutant
Jack J.-N. Liang
Center for Ophthalmic Research/Surgery, Brigham and Women's Hospital, Boston, MA; Department of Ophthalmology, Harvard Medical School, Boston, MA
Correspondence to: Jack Liang, Center for Ophthalmic Research/Surgery, 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
Purpose: Missense mutations in crystallin genes have been identified in autosomal dominant congenital cataracts. A truncation in the CRYBB2 gene (Q155*) has been associated with cerulean cataract, however its effects on biophysical properties have not been reported. We sought to determine the changes in conformation and protein-protein interactions brought about by this mutation.
Methods: Site specific mutations were performed to obtain the Q155* βB2-crystallin mutant. Protein-protein interactions were screened by a mammalian two-hybrid system assay. Conformational changes were studied with spectroscopy (circular dichroism and fluorescence) and FPLC chromatography.
Results: We detected a decrease in protein-protein interactions for the Q155* βB2-crystallin mutant. The Q155* mutant shows decreased ordered structure and stability but the partially unfolded protein retains some dimer structure.
Conclusions: The Q155* mutation in βB2-crystallin causes changes in biophysical properties that might contribute to cataract formation.
β-Crystallin is one of three major lens crystallin components (α-, β-, and γ-crystallin) and is further subdivided into acidic (βA1, βA2, βA3, and βA4) and basic (βB1, βB2, and βB3) components . They form heterogeneous oligomers in the lens and have molecular weights ranging from 40 to 200 kDa. βB2-crystallin is the major β-crystallin component and is a dimer at low protein concentrations [2,3]. The structure of βB2-crystallin has been reported [4-7] and is very similar to that of γB-crystallin , except that the link peptide is extended in βB2-crystallin. The β- and γ-crystallin are now considered to belong to the β/γ superfamily. The known structures for β- and γ-crystallin greatly facilitate the study of conformational changes caused by either posttranslational modifications or mutations.
β- And γ-crystallins were thought to be specific to lens fiber cells, but recently it was reported that some β- and γ-crystallin components were found in lens epithelial cells . Besides serving as structural proteins and refractive index gradients, their functions in the lens are not known. Like α-crystallin but to a lesser extent, β- and γ-crystallin are subjected to posttranslational modifications and progressively become aggregated and insoluble with age, leading to age related cataract formation. In a change similar to posttranslational modification, site specific mutations in crystallins also cause conformational changes and aggregation, as seen in autosomal dominant congenital cataracts [10-12].
The mutant gene CRYBB2 (Q155*) has been reported to cause the cerulean cataract, one of many autosomal dominant congenital cataracts . The Q155* truncation eliminates 51 amino acids in the C-terminal region and must exert great effects on protein conformation, stability, and interaction properties. Other congenital cataract genes, such as CRYAA (R116C) in zonular central nuclear cataract , CRYAB (R120G) in desmin related myopathy , and CRYGC (T5P) in Coppock-like cataract  have been extensively studied [10-12,17,18]. For example, we have studied the effects of the T5P mutation on γC-crystallin; decreased protein-protein interactions and conformational changes were observed [12,19]. It will be interesting to see the effects of the Q155* mutation on protein-protein interactions and other biophysical properties.
The Clontech Mammalian Two-Hybrid System Assay 2 Kit (Clontech, Palo Alto, CA) was used. The test protein (bait) was fused into the GAL4 DNA-BD in the pM vector and the second test protein (prey) was fused into the VP16-AD in the pVP16 vector. The third vector pG5SEAP contains a reporter construct and is encoded with secreted alkaline phosphatase (SEAP), an enzyme that enables one to sample cultured medium without cell lysis.
Using previously made plasmids, pM-βB2 and pVP16-βB2 , the Q155* mutants (pM-Q155* and pVP16-Q155*) were prepared using the Quick-Change Mutagenesis kit (Stratagene, La Jolla, CA). The primers used were custom synthesized (Invitrogen): the forward primer was GTG GGT TGG CTA CTA GTA CCC CGG CTA CC and the reverse primer was GGT AGC CGG GGT ACT AGT AGC CAA CCC AC. The other crystallin inserts (pM-αA, pVP16-αA, etc) had been previously prepared [19,20].
Cotransfection and SEAP assay
HeLa cells were used for cell culture and Lipofectamine 2000 (Invitrogen, Rockville, MD) was used for cotransfection. HeLa cells were grown at 37 °C with 5% CO2 with 10% serum and were seeded at 2x105 cells in 500 μl medium per well in 24 well plates. Plasmids pM-X (0.3 μg) and pVP16-Y (0.3 μg), and the pG5SEAP reporter vector (0.3 μg) were added to the wells containing 2 μl of Lipofectamine 2000 reagent.
SEAP activity was measured 48 h after transfection using the BD Great EscAPe SEAP Fluorescence Detection Kit (Clontech). The SEAP reporter gene encodes a truncated form of the placental enzyme without the membrane anchoring domain, allowing the protein to be efficiently secreted from transfected cells. It was reported that levels of SEAP activity detected in the culture medium were directly proportional to changes in intracellular concentrations of SEAP mRNA and protein . The detailed protocol for SEAP detection is provided in the kit. The fluorescence substrate, MUP (4-methylumbelliferyl phosphate), provides an easy assay of SEAP by fluorescence reading at 360/449 nm. The readings were normalized with the readings of the controls (cotransfection of pM + pVP16).
Expression of the Q155* βB2-crystallin mutant
Initial results of expression experiments in bacterial cell culture indicated a low yield for the Q155* mutant, which hampered purification. To facilitate expression and purification, we used 6xHis tagging for protein expression in bacterial cells. The Trp fluorescence and CD measurements indicated no conformational change in the tagged protein compared to the untagged protein. The tagging greatly reduced the time and procedure for purification.
The QIAexpression Type IV kit was used for cloning, expression, and purification (Qiagen, Valencia, CA). The pQE-30 vector contained 6xHis-tag coding sequence in the N-terminal region, which facilitates binding of expressed protein to agarose coupled nickel-nitrilotriacetice acid (Ni-NTA). The βB2-crystallin genes in pM plasmids (pM-βB2WT [wild type] and pM-βB2Q155*) were amplified by PCR using Pfu DNA polymerase (Stratagene) with the forward/reverse primers: CGG GGT ACC CCG GCC TCA GAT CAC CAG/CCC AAG CTT GGG GTT GGA GGG GTG GAA for the WT βB2-crystallin and CGG GGT ACC CCG GCC TCA GAT CAC CAG/CCC AAG CTT GGG GTA GCC AAC CCA CGT for the Q155* mutant. Two restriction sites, KpnI and HindIII, were included in the primers. The PCR products and pQE-30 vector were doubly digested by KpnI and HindIII. The digested genes and vector were then ligated by DNA ligase under standard conditions. The βB2-crystallin cDNA inserts were verified by sequence analysis.
The expression constructs containing βB2-crystallin genes were transformed into E. coli strain M15 [pREP4]. Cell culture was performed to induce protein expression using standard protocol.
The 6xHis tagged βB2-crystallins, either WT or Q155* mutant, were purified by Ni-NTA affinity chromatography, which simplifies purification. The 6xHis tagged βB2-crystallin was compared with the previously prepared untagged βB2-crystallin by CD and Trp fluorescence and the results confirmed the common perception that the 6xHis tagging does not interfere with the structure/function of the recombinant proteins.
Study of biophysical properties
SDS-PAGE was performed in a slab gel (15% acrylamide) under reducing conditions according to the method of Laemmli . Western blotting was performed with polyclonal anti β-crystallin antibodies (a gift from Dr. Sam Zigler). Protein concentrations were determined by measuring absorption at 280 nm (A0.1%=1.74 for WT and 1.70 for Q155* mutant based on the amount of Trp and Tyr residues) .
Size exclusion chromatography was carried out using FPLC equipped with FPLCdirector software using a superose-12 column (Pharmacia, Piscataway, NJ).
CD spectra were obtained with an Aviv Circular Dichroism Spectrometer (model 60 DS; Aviv Associates, Lakewood, NJ). Five scans were recorded, averaged, and followed by a polynomial fitting program. The CD was expressed with a unit of deg-cm2/dmol.
Fluorescence was measured with a Shimadzu spectrofluorometer (model RF-5301PC; Shimadzu Instruments, Columbia, MD). Trp emission was scanned with an excitation wavelength of 295 nm. Bis-ANS fluorescence emission spectra were scanned between 460 and 560 nm with an excitation wavelength of 395 nm. Aliquots of 50 μl of Bis-ANS (5.5x10-5 M stock solution) were added to 1 ml of βB2-crystallin solution (0.08 mg/ml) until saturation.
For stability measurements, Trp fluorescence was measured for samples in guanidine hydrochloride (GdHCl). The samples of either WT or Q155* βB2-crystallin in increasing concentrations of GdHCl were incubated overnight at room temperature. The Trp fluorescence was scanned with λex at 295 nm.
Protein-protein interactions involving βB2-crystallin mutants
The SEAP activity as detected by MUP fluorescence between βB2-crystallins themselves is shown in Figure 1. There was a more than 20 fold increase in SEAP activity with cotransfection of (pM-X + pVP16-Y) for WT βB2-crystallin compared with the negative controls (pM + pVP16); the Q155* mutation decreased SEAP activity significantly in the cotransfections of (pM-Q155* + pVP16-Q155*) and (pM-Q155* + pVP16-WT; p=0.003). A previous study indicates that βB2-crystallin shows rather weak but detectable interactions with αA-, αB-, and γC-crystallins  and that mutations in these crystallins change the interactions, either decreasing for R116C αA-crystallin and T5P γC-crystallin or increasing for R120G αB-crystallin . However, the Q155* mutation of βB2-crystallin showed no significant change in interactions with other crystallins (pM-αA + pVP16-Q155*, pM-αB + pVP16-Q155*, and pM-γC + pVP16-Q155*; p>0.05, Figure 2). To determine whether protein concentrations varied among the cotransfections, cultured cells were lysed and protein concentrations were determined. The results indicated very little variation. The change in SEAP activity was not caused by different protein expression. In the initial experiments, a second reporter vector (pEGFP-C1) encoding a gene of enhanced green fluorescent protein (EGFP) was included in the cotransfection. The EGFP fluorescence levels of cell lysates were measured at 489/509 nm, and no difference in the transfection efficiency among various cotransfection experiments was observed.
Biophysical studies of βB2-crystallin mutants
The His tagged WT βB2-crystallin and Q155* mutant were prepared. The 6xHis tagging did not change protein conformation as demonstrated by the lack of difference in Trp fluorescence and CD between the His tagged and the untagged WT βB2-crystallin (see CD and Trp fluorescence results).
SDS-PAGE, western blot, and size exclusion chromatography
Figure 3A shows the SDS-PAGE for the Q155* βB2-crystallin mutant. The Q155* mutant band is located at a lower molecular weight than the WT band, indicating a truncation of 51 amino acids (5,865 Da). The western blot band for the Q155* mutant appeared less stained than the WT (Figure 3B), possibly because of removal of some epitope sites or weak epitope affinity in the mutant.
FPLC data indicated that the Q155* mutant had a smaller molecular size than the WT (Figure 4). βB2-crystallin is a dimer with molecular size of 46 kDa . The Q155* mutant has an expected size of 35 kDa but its distribution profile is wider than that of the WT and extends to the position of monomeric γC-crystallin. The truncation did not inhibit the dimerization but the dimerization was not complete; the dimer and monomer were in equilibrium.
CD data for the Q155* mutant are included in Figure 5. Both far UV and near UV CD spectra were significantly different from those of WT, indicating changes not only in secondary structure but also tertiary structure. Using the Prosec program, the secondary structural content (α-helix, β-sheet, β-turn, and random coil) for Q155* mutant is 5%, 27%, 32%, and 35% while the corresponding content for WT is 11%, 39%, 22%, and 28%, respectively.
Trp and Bis-ANS fluorescence
Trp emission maximal wavelength for the Q155* mutant showed a red shift from 331 nm to 340 nm and a more than two fold decrease in intensity, indicating that the Trp residues were in a more hydrophilic environment in the mutant than in the WT βB2-crystallin (Figure 6).
Bis-ANS fluorescence also showed great changes in both intensity and emission maximal wavelength: a shift of maximal emission wavelength from 499 nm to 492 nm and a more than two fold increase in intensity (Figure 7).
GdHCl unfolding study
The fluorescence intensity ratios at 360 nm and 320 nm (I360/I320) or maximal emission wavelengths were plotted against GdHCl concentrations (Figure 8) . While the WT βB2-crystallin showed a sigmoid unfolding curve, the mutant showed a hyperbola unfolding curve. The unfolding curve for the mutant was shifted to lower GdHCl concentrations indicating that the mutant had a lower stability than the WT. Since only the unfolding experiment was carried out, the results provided the rank of stability rather than the thermodynamic stability.
The SEAP activity measurements show that a truncation of 51 amino acids in the C-terminal region of βB2-crystallin reduced the activity but did not eliminate the protein-protein interaction completely. The dimerization of βB2-crystallin arises from an intermolecular association similar to the intramolecular interaction observed in γ-crystallin. The interaction sites are believed to be β-sheets, each composed of two or more β-strands. We can take a closer look at the structural basis for the change of the activity. Although the x-ray crystallographic structure has been reported for the dimeric βB2-crystallin , the protein studied was a recombinant fragment of 181 amino acids. It would be very informative to compare βB2-crystallin with a monomeric γB-crystallin, since they have a high homology. The crystallographic structure of γB-crystallin has been well characterized; it consists of four Greek key motifs, two motifs (1 and 2) in the N-terminal domain and two motifs (3 and 4) in the C-terminal domain . The two domains are connected by a flexible linker peptide that favors an intra molecular domain association. β-Crystallin has a structure similar to γ-crystallin except that the linker peptide that is extended favors an inter molecular association. The sequence alignment between βB2- and γB-crystallin is shown in Figure 9. The distribution of β-strands are very similar in these two crystallins; both have 14 β-strands. γB-crystallin has two additional α-helices but βB2-crystallin has none. Based on the x-ray crystallography structure, the Q155* mutation removes the last three β-strands in the sequence.Our preliminary results in the determination of the interaction domains of βB2-crystallin confirm that the last three β-strands of the C-terminal domain are partially involved in dimerization (unpublished). FPLC clearly indicates that the Q155* mutant is a dimer or is in monomer-dimer equilibrium. The incomplete dimerization must be caused by changes in secondary and tertiary structures.
The spectroscopic results indicate that truncation affects normal folding; the protein is partially unfolded, as evidenced by Trp fluorescence and CD data. The βB2-crystallin has 5 Trp residues (W-59, W-82, W-85, W-151, and W-195). The Q155* mutation removed only one Trp residue (W-195), this cannot account for the large change in Trp fluorescence. The mutation has changed not only the tertiary structure but also the secondary structure as shown by the CD results. The reduced secondary structural content disrupts the tertiary structure, resulting in partial unfolding. The loosening of the structure is also indicated by the Bis-ANS fluorescence, which shows that more hydrophobic surfaces are exposed in the Q155* mutant than in the WT βB2-crystallin. The partial unfolding of the βB2-crystallin was also demonstrated by the unfolding curve with GdHCl, in which the partially unfolded protein was further unfolded at lower GdHCl concentrations than the WT βB2-crystallin.
The effects of the Q155* mutation at physiological concentrations such as those found in the lens will become more prominent than in dilute solutions. βB2-crystallin is a dimer at low concentrations but becomes an oligomer, either homo- or hetero-oligomeric, at high concentrations. At such high concentrations, the Q155* mutant will have far less normal protein-protein interactions than the WT, but will have greater hydrophobic interactions that promote HMW aggregation and insolubilization. The exposure of hydrophobic surfaces facilitates the hydrophobic interactions that decrease protein solubility.
Like mutations of R116C αA-, R120G αB-, and T5P γC-crystallin, the Q155* mutation of βB2-crystallin is another example of human cataract caused by conformational change and decreased stability [10-12]. It is generally believed that, while congenital cataracts are caused by single site specific mutations, age related cataracts are brought about by multiple posttranslational modifications, which may occur at many sites in various crystallins. Therefore, congenital cataract crystallin mutation provides a better model in which to study the relationship between protein alteration and cataractogenesis.
This work was supported by grants from the National Institutes of Health (EY13968) and Massachusetts Lions Eye Research Fund.
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