(Received for publication, November 18, 1996, and in revised form, December 17, 1996)
From the Center for Ophthalmic Research, Brigham and Women's Hospital, and the Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02115
Human and other mammalian lens proteins are
composed of three major crystallins: -,
-, and
-crystallin.
-Crystallin plays a prominent role in the supramolecular assembly
required to maintain lens transparency. With age, the crystallins,
especially
-crystallin, undergo posttranslational modifications that
may disrupt the supramolecular assembly, and the lens becomes
susceptible to other stresses resulting in cataract formation. Because
these modifications occur even at a relatively young age, it is
difficult to obtain pure, unmodified crystallins for in
vitro experiments.
-Crystallin is composed of two subunits,
A and
B. Before the application of recombinant DNA technology,
these two
-crystallin subunits were separated from calf lens in the
denatured state and reconstituted by the removal of the denaturant, but
they were not refolded properly. In the present studies, we applied the
recombinant DNA technology to prepare native, unmodified
A- and
B-crystallins for conformational and functional studies. The
expressed proteins from Escherichia coli are in the native
state and can be studied directly. First,
A and
B cDNAs were
isolated from a human lens epithelial cell cDNA library. The
cDNAs were cloned into a pAED4 expression vector and then expressed
in E. coli strain BL21(DE3). Pure recombinant
A- and
B-crystallins were obtained after purification by gel filtration and
DEAE liquid chromatography. They were subjected to conformational
studies involving various spectroscopic measurements and an assessment
of chaperone-like activity.
A- and
B-crystallins have not only
different secondary structure, but also tertiary structure.
1-Anilino-8-naphthalene sulfonate fluorescence indicates that
B-crystallin is more hydrophobic than
A-crystallin. The chaperone-like activity, as measured by the ability to protect insulin
aggregation, is about 4 times greater for
B- than for
A-crystallin. The resulting data provide a base line for further studies of human lens
-crystallin.
-Crystallin is the major lens structural protein responsible
for the supramolecular assembly necessary to maintain lens
transparency. It is isolated as a large water-soluble aggregate with a
mass of 800 kDa and is composed of two homologous subunits,
A and
B, each with a molecular mass of 20 kDa. With aging and cataract formation,
-crystallins gradually become a high molecular weight aggregate and eventually an insoluble protein (1). This is manifested
by the observation that the major crystallin component in the insoluble
protein fraction of aged and cataractous lenses is
-crystallin (2).
The aggregation and insolubilization of
-crystallin are believed to
arise from posttranslational modifications, which accumulate
significantly because of the low turnover rate of lens crystallins.
Two-dimensional gel electrophoresis indicated that human lens
crystallins were modified extensively even at a relatively young age
(3). The significance of the modification is that crystallins become
partially unfolded, and this partially unfolding exposes hydrophobic
surfaces and promotes hydrophobic interaction. In aged and cataractous
lenses, aggregation and insolubilization take place because the
concentration of partially unfolded protein is high.
Currently, most human lens specimens available for in vitro
studies are from old human subjects or cataract surgery, and the crystallins, especially -crystallin isolated from these specimens, are extensively modified. Therefore, it is difficult to obtain pure and
unmodified human lens
-crystallin for in vitro
investigations. An alternative is to use an animal model, but this
option raises concern about whether the results are species-specific or
can be extrapolated to humans. For these reasons, many investigators used recombinant DNA technology to clone lens
-crystallin (4-11). The technique was employed to prepare not only unmodified
-crystallin but also site-specific mutants to study the effect of
site-specific mutation on protein conformation and function. These
studies, however, dealt only with either
A-crystallin (4-8, 10, 11) or
B-crystallin (9). Moreover, no comparative study on conformation and function between recombinant
A- and
B-crystallin has been reported. In the present study, we prepared both recombinant human
A- and
B-crystallins and characterized their conformations by molecular spectroscopy. The results provide a base line for further studies of modification-related changes in protein interaction and
conformation. In addition,
A- and
B-crystallins were recently detected in other tissues, such as heart, kidney, spleen, and retina
(12-15). These findings raise question about the role or function of
A- and
B-crystallins in these tissues. Similar studies of the
corresponding recombinant
A- and
B-crystallins from tissues other
than the lens may yield information on their conformation and
aggregation state. This information may provide a clue to their
biological functions.
A complementary DNA library of HLE cells prepared with a
ZAP-cDNA Synthesis Kit (Stratagene, La Jolla, CA)
was generously provided by Dr. Toshimichi Shinohara. The A gene (689 bp)1 and the
B gene (657 bp) were isolated by PCR using
two specific primers for each. The forward primer,
d(AAGCTG
GACGTGACCATCCAGCACCC) for
A and
d(CTAGCC
GACATCGCCATCCACC) for
B, corresponds to a
cDNA sequence containing an ATG translation start codon and incorporating a two-base change (AACATG
CATATG) for
A and a three-base change (ACCATG
CATATG) for
B to create an
NdeI restriction site. The reverse primer for
A,
d(GAC
CTTTCCTGGCTGCTCTCTCAAACC), corresponds to the
cDNA sequence 125 nucleotides downstream of the AAG stop codon and
incorporates a HindIII site. The reverse primer for
B,
d(TATTAGCTTGATAATTTGGGCCTGCCCTTAGC), corresponds to the cDNA
sequence 89 nucleotides downstream of the UAG stop codon. The two
restriction sites were created for the purpose of cloning
A or
B
cDNA into the T7 expression vector pAED4, a 3322-bp
ampicillin-resistant plasmid containing the restriction sites
NdeI and HindIII at the polycloning sites (16).
The primers were custom-synthesized by Life Technologies, Inc.
For PCR, we used a protocol for Pfu DNA polymerase supplied
by the manufacture (Stratagene). The PCR mixture (10 ng of human lens
epithelial cDNA, 200 pmol of each amplification primer, 10 µl of
10 × Pfu DNA polymerase buffer, 20 nmol each of four
dNTPs, and 2.5 units of Pfu DNA polymerase in a final volume
of 100 µl) was first heated to 95 °C for 1 min to denature the
template DNA; it was then subjected to 30 cycles of 95 °C (45 s),
55 °C (1 min), and 72 °C (2 min) each. The PCR product was
purified by 2% agarose gel electrophoresis and was double-digested
with NdeI and HindIII. The expression vector
pAED4 was similarly double-digested. The digested gene and vector were
then ligated by DNA ligase. Escherichia coli strain NM522,
which lacks the gene for T7 RNA polymerase, was transformed with 10 µl of ligation mixture. The transformed cells were spread on agarose
plates containing ampicillin (100 µg/ml), and 10 clones were picked
out. The expression construct was screened by double digestion with
NdeI and HindIII to confirm the presence of the
cDNA insert. The expression construct was propagated in E. coli strain NM522. The nucleotide sequence of the A or
B
cDNA in the construct was determined by Sanger sequencing; a
fluorescence-tagged dideoxy termination method was used with an ABI
Automatic Sequencing System (Brigham and Women's Hospital Automatic
Sequencing and Genotyping Facility).
For the overexpression of A- and
B-crystallins, E. coli strain BL21(DE3) was transformed
with expression constructs pAED4-
A and pAED4-
B, respectively. A
colony was picked out from the freshly streaked plate and inoculated
with 50 ml of LB containing ampicillin (50 µg/ml). The culture was
grown to log phase (A590, 0.6-0.9). A 20-ml
volume of the log phase culture was inoculated into 1 liter of LB
containing ampicillin (50 µg/ml). The culture was incubated at
37 °C with vigorous shaking until the optical density reached
0.6-0.9 at 590 nm, at which point expression was induced by the
addition of 1 ml of 1 M isopropyl
-D-thiogalactoside. Incubation was continued for another
3 h. Cells were then harvested by centrifugation at 5,000 rpm for
10 min and kept at
80 °C. A control culture of E. coli
BL21(DE3) cells with pAED4 was incubated and induced
simultaneously.
The collected cells were suspended in TNE buffer (50 mM
Tris-HCl, 1 mM EDTA, 100 mM NaCl) at a ratio of
3 ml of buffer to 1 g (wet weight) of cells, and 8 µl of 50 mM phenylmethylsulfonyl fluoride and 80 µl of lysozyme
(10 mg/ml) per g of cells were added. The mixtures were kept at 4 °C
for 20 min, after which deoxycholic acid (4 mg/g of cells) was added.
After thorough mixing, DNase (1 mg/ml, 20 µl/g of E. coli
cells) was added, and incubation at 37 °C was continued until the
mixture was no longer viscous. By centrifugation at 12,000 rpm for 30 min, the cell lysates were separated into supernatant and insoluble
pellets. The recombinant proteins were purified from the supernatant by
the following successive steps: fractionation with 30-60% saturated
ammonium sulfate, gel filtration on Sephacryl S-300HR, and ion exchange
chromatography on DEAE-Sephacel. The purity of the recombinant A-
and
B-crystallins was confirmed with FPLC gel filtration, SDS-PAGE,
and isoelectric focusing (IEF) gel electrophoresis, and the crystallin
preparations were stored in 0.05 M phosphate buffer at
20 °C.
FPLC was carried out on a Superose-6 column (Pharmacia Biotech Inc. FPLC System) (17). The sample solutions were passed through a filter (pore size, 0.45 µm) before application to the column. The column was eluted at 0.3 ml/min. Gel filtration standards from Pharmacia were used for calibration.
SDS-PAGE and Western BlotSDS-PAGE was performed in a slab gel (15% acrylamide) by the method of Laemmli (18) under reducing conditions. Electrophoresis was conducted with a constant voltage of 200 V for 1 h. The gels were stained with Coomassie Brilliant Blue R-250.
Proteins separated by SDS-PAGE were electrophoretically transferred to a nitrocellulose membrane with the Trans-Blot Transfer Cell (Bio-Rad). Transfer was carried out with low field current (30 V and 0.01 A) overnight. The detailed procedures were supplied by the manufacturer (Bio-Rad).
IEFIEF was performed on the precast IsoGel agarose IEF gels (pH range, 3-10; FMC BioProducts, Rockland, ME) under either native or denatured conditions. Gels were stained with Coomassie Brilliant Blue R-250.
CD MeasurementsCD spectra were measured with an Aviv Circular Dichroism Spectrometer (model 60 DS) (19). The reported CD spectra are the average of 5-10 scans, smoothed by polynomial curve fitting. The fit was checked with a statistical test so that the original data were not oversmoothed. The CD data were expressed as molar ellipticity in degrees cm2/dmol, with 115 as residue molecular weight.
Fluorescence MeasurementsFluorescence measurements were
performed with a Shimadzu spectrofluorometer (model RF-5301PC). Trp
fluorescence was measured with an excitation wavelength at 295 nm. The
fluorescent probe, ANS, was used to study the hydrophobicity of protein
molecules (20). The ANS (~50 µM) in -crystallin (0.1 mg/ml in 0.05 M phosphate buffer, pH 7.6) was incubated at
room temperature for 30 min before the measurements were made. The
emission was measured with an excitation wavelength at 395 nm.
Chaperone activity was
assayed by aggregation of insulin. Aggregation was induced by
dithiothreitol reduction of insulin B-chain in the presence and absence
of -crystallin. The procedures were similar to those reported by
Farahbakhsh et al. (21), except that samples were prepared
in a 96-well microplate. In brief, 0.4 mg of insulin was reduced with
10 mM dithiothreitol in the presence or absence of
-crystallin samples (0.05-1.6 mg) in a final volume of 250 µl.
The microplate was read at 490 nm with a Bio-Tek EL-800 microplate
reader at room temperature. The reading was recorded every 3 min until
a plateau was reached. Observed optical densities were recorded and
analyzed with the KC3 software provided by Bio-Tek.
Protein concentrations were determined with a Pierce bicinchoninic acid (BCA) assay (22).
A- and
B-crystallin cDNAs amplified by PCR
were analyzed on 2% agarose gel;
A cDNA (689 bp) showed a
0.7-kb band and
B cDNA (657 bp) showed a 0.65-kb band. After
cloning into pAED4 and double digestion with NdeI and
HindIII, the 1% agarose gel displayed a 3.3-kb band
(3322-bp vector) and a 0.68-kb band (670-bp
A cDNA insert) for
pAED4-
A and a 3.3-kb band (3322-bp vector) and a 0.60-kb band
(607-bp
B cDNA insert) for pAED4-
B. A single digestion of
pAED4-
A and pAED4-
B showed a single band of 4.0 kb (3322-bp
vector plus insert). DNA sequencing of the 5
-end sequence of
A and
B cDNA in the expression constructs confirmed the correct
reading frame.
Human lens A-crystallin (173 residues) and
B-crystallin (175 residues) were expressed in host E. coli BL21(DE3) using pAED4 expression plasmid. The isopropyl
-D-thiogalactoside induction of pAED4-
A and
pAED4-
B resulted in the expression of a polypeptide with an apparent
molecular mass of 20 kDa, corresponding to the molecular mass of the
A and
B subunits (Fig. 1, lanes 2 and 4). After cell lysis, the water-soluble fractions of the
lysates were used to purify recombinant
A-crystallin and
B-crystallins. The purified products were seen as a single band in
SDS-PAGE and IEF gels (Figs. 1 and 2). Western blotting
identified the
A-crystallin, but not
B-crystallin, with
polyclonal anti-
antiserum (Fig. 3). This result may
have been due to the low immunoaffinity for
B, as reported by
Thomson and Augusteyn (23). The expression yield was about 30% of
total bacterial protein, as estimated by FPLC profiles.
A- and
B-crystallins have almost the same apparent molecular mass, which is
slightly lower than that of calf
-crystallin (700-800 kDa) (Fig.
4). This result indicates that recombinant
A and
B
are properly folded and aggregated in E. coli. Their slightly smaller size may be due to the absence of modifications.
CD Measurements
The near-UV CD spectra of recombinant A-
and
B-crystallins are very different (Fig. 5). The
difference may not arise solely from the different content of aromatic
amino acids (Trp, Tyr, and Phe) but probably also involves a different
tertiary structure; CD intensity is related more to the rotational
strength of these residues than to their amount, and the rotational
strength in turn depends on the compactness of the protein structure
(24).
The far-UV CD spectra are shown in Fig. 6;
B-crystallin displays a CD quite different from that of
A-crystallin. For
A-crystallin, the typical 218-nm band for
-sheet conformation is apparent, and the position is shifted to a
shorter wavelength for
B-crystallin. For a more precise estimate of
the percentage of various secondary structures, we used the computer
program SELCON provided by Greenfield (25), which is based on the work
of Johnson (26) and Sreerama and Woody (27). The program utilized CD
spectra of proteins for which the secondary structures had been
determined by x-ray diffraction. Three structural components
(
-helix,
-sheet, and
-turn) were obtained, with the remaining
structure considered a random coil. The percentage of each secondary
structure in this order is 5, 40, 16, and 39% for
A-crystallin and
14, 33, 18, and 35% for
B-crystallin. The figures agree well with
previous reports that the major secondary structure of
-crystallin
is
-conformation (50-60%,
-sheet and
-turn combined) and
that there is very little
-helical content (~5%) (28, 29);
however, the present study indicates that
B-crystallin has a greater
content of
-helix than
A-crystallin does.
Trp and ANS Fluorescence
The Trp emission maximum provides a
direct indication of whether the Trp residues (Trp-9 in A-crystallin
and Trp-9 and Trp-60 in
B-crystallin) are buried or exposed. Since
all of these residues are all in the N-terminal domain (residues
1-63), which is hydrophobic and participates in high molecular weight
aggregation, they should be relatively buried.
A- and
B-crystallin display emission maxima at 335 and 337 nm,
respectively, which are close to that for calf
-crystallin (336 nm)
(Fig. 7).
ANS is a hydrophobic probe. Upon binding to protein hydrophobic
surfaces, ANS fluorescence intensity increases, and the emission maximum shifts to a shorter wavelength, thus providing a sensitive measurement for probing protein hydrophobicity. Fig. 8
shows that ANS fluorescence is more than 3 times greater in B- than
in
A-crystallin, a difference indicating that
B- is more
hydrophobic than
A-crystallin.
Chaperone-like Activity
The protection of insulin from
aggregation by calf -crystallin has been extensively studied (21).
We included calf
-crystallin for comparison in our study of
recombinant
A and
B (Fig. 9). Clearly,
B-crystallin is far more effective than
A-crystallin in
preventing insulin aggregation. In the protocol described under "Materials and Methods," 0.2 mg of
B-crystallin can completely protect insulin from aggregation, whereas 1.6 mg of
A-crystallin is
required. Chaperone-like activity thus appears to correlate directly
with protein hydrophobicity.
Employing the DNA cloning technology, we have prepared pure A-
and
B-crystallins. The aggregation sizes of the pure preparations are slightly smaller than that of
-crystallin isolated from calf lens water-soluble fraction. It is not known whether this difference is
due to the lack of modifications during folding and aggregation in
E. coli, but
-crystallin does increase in size with
development and aging. Conformational studies indicate that
A- and
B-crystallins have an amount of
-conformation comparable with
that of calf lens
-crystallin but that
B-crystallin has
substantially more
-helical content, whose significance is not
clear. The difference in tertiary structure, as manifested by the
difference in near-UV CD (Fig. 5), may contribute to conformational
stability. To our knowledge, no study of the difference in
conformational stability between recombinant
A- and
B-crystallin
has been reported.
The most striking difference between A- and
B-crystallin is the
greater hydrophobicity and thus the greater chaperone-like activity for
B- than for
A-crystallin. The significance of this finding is
related to the facts that
B-crystallin is expressed in more tissues
than
A-crystallin (13), that
B-crystallin is expressed at higher
levels than
A-crystallin in some tissues (30, 31), and that
B-crystallin, like the small heat-shock protein, may function as a
stress protein and may be expressed under various stress conditions and
diseases (32-34). The greater chaperone function of
B-crystallin
thus effectively stabilizes other proteins under stress and confers
thermostability to cells. The mechanism for the chaperone-like activity
of
-crystallin is interaction with other proteins partially unfolded
by heat, denaturant, or other mechanisms such as the reduction of
insulin (21, 35, 36).
The A and
B subunits were conventionally separated under
denatured conditions (e.g. in urea or guanidine HCl), and
the
A- and
B-crystallins were reconstituted by removal of the
denaturant (23, 37-39). The molecular weights of these reconstituted
-crystallins were always lower than that of native
-crystallin,
indicating that the reaggregation is incomplete. The recombinant
A- and
B-crystallins thus provide the advantage that they are in
the native form, which can be important to studies of the dynamic quaternary structure of
-crystallin. Previous studies with
renatured
-crystallin (homopolymer and heteropolymer) indicated that
the
A and
B subunits are structurally equivalent and
interchangeable (23, 39). Such studies using recombinant
A- and
B-crystallins may yield more relevant data, since in vivo
A and
B subunits are interchanged under the native conditions
after
A- and
B-crystallins are expressed and folded.
The biological significance of the 3:1 ratio of A to
B subunits
in the mammalian
-crystallin has not been established, although
previous reports indicated that any ratios can be formed in
vitro (23, 39). It is believed that the significance of the
A:
B ratio may be implied by the result of a comparative study on
the conformational stability of
-crystallins formed by various
ratios of
A and
B subunits. It is possible that
-crystallin with a 3:1 ratio of
A to
B is the most stable form.
A recent chaperone study used a site-specific mutation on
A-crystallin to study conformational change of the substrate bound to
-crystallin (40). A Trp-free
A-crystallin mutant was prepared, and Trp fluorescence of the substrate protein was probed during the
chaperone action. The results, along with those of many previous recombinant studies (4-11), demonstrated the versatility of the recombinant DNA technology. During cataract formation, many
posttranslational modifications, including nonenzymatic glycation,
protein-protein and mixed disulfide formation, and photooxidation of
Trp residues, were observed. Use of site-specific
-crystallin
mutants may help us to understand modification-induced aggregation and
insolubilization. For example, use of a Trp-free
-crystallin mutant
in the UV irradiation studies may shed light on the role of Trp
residues in lens damage due to sunlight. This and other studies
mentioned above are under way in our laboratory.
We are grateful to Dr. Toshimichi Shinohara for the gift of human epithelial cell cDNA library and for helpful discussion during the course of this study.