From the Center for Ophthalmic Research, Brigham and Women's Hospital, and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
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Lens -crystallin subunits
A and
B are
differentially expressed and have a 3-to-1 ratio in most mammalian
lenses by intermolecular exchange. The biological significance of this
composition and the mechanism of exchange are not clear. Preparations
of human recombinant
A- and
B-crystallins provide a good system
in which to study this phenomenon. Both recombinant
A- and
B-crystallins are folded and aggregated to the size of the native
-crystallin. During incubation together, they undergo an
intermolecular exchange as shown by native isoelectric focusing.
Circular dichroism measurements indicate that the protein with a 3-to-1
ratio of
A- and
B-crystallins has the same secondary structure
but somewhat different tertiary structures after exchange: the near-UV
CD increases after exchange. The resulting hybrid aggregate is more
stable than the individual homogeneous aggregates: at 62 °C,
B-crystallin is more susceptible to aggregation and displays a
greater light scattering than
A-crystallin. This heat-induced
aggregation of
B-crystallin, however, was suppressed by
intermolecular exchange with
A-crystallin. These phenomena are also
observed by fast performance liquid chromatography gel filtration
patterns. The protein structure of
B-crystallin is stabilized by
intermolecular exchange with
A-crystallin.
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INTRODUCTION |
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-Crystallin is an oligomer protein of two subunits,
A and
B, in a ratio of 3 to 1 for most mammalian lenses. The
A- and
B-crystallins are differentially expressed in the lenses of humans and most other mammalian species;
B-crystallin appears earlier than
A-crystallin and is dominant in epithelial cells, but the
A-to-
B ratio increases with differentiation (1-4). The
significance of the differential expression is not clear.
Moreover, how the two subunits interchange after expression and
the significance of the 3-to-1 ratio are also not well established.
The rather high concentration of -crystallin in the lens and its
extremely high thermostability (5) may be crucial in the protection
against aggregation of
- and
-crystallins. But how the
thermostability is achieved is not fully understood. One possibility is
through an intermolecular exchange that results in a dynamic quaternary
structure. The observation that
-crystallin is the major crystallin
in the lens HMW1 aggregates
and water-insoluble fraction (6) may arise from some mechanisms that
cause the loss of this dynamic quaternary structure.
In vitro study of -crystallin has been handicapped by the
fact that
-crystallin and its two subunits, especially isolated from
human lenses, are already extensively modified and are difficult to
obtain in their pure, native state. Previously, the two subunits were
isolated from
-crystallin under denatured conditions and were unable
to refold to the native state. The use of recombinant DNA technology to
clone human
-crystallin has largely solved these problems (7-10).
We have recently prepared both recombinant human
A- and
B-crystallins and performed a comparative study between them (10).
The recombinant
A- and
B-crystallins exist in solution as
homo-oligomers of molecular mass ~600 kDa (10). In this report, we
present evidence of the interoligomeric exchange of the
A- and
B-crystallins and the stabilization of protein structure by subunit
interchange. The biological significance of the 3-to-1 ratio of
A
and
B subunits in vivo is discussed.
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EXPERIMENTAL PROCEDURES |
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Materials--
Recombinant human A- and
B-crystallins were
prepared from a cDNA library of human lens epithelial cells, kindly
provided by Dr. Toshimich Shinohara. The procedures for cloning,
expression, and protein purification have been described previously
(10). Proteins in 0.05 M phosphate buffer, pH 7.4, were
used in all measurements unless otherwise stated. Protein
concentrations were determined by calculated extinction coefficients
based on a protein amino acid sequence as described by Mach et
al. (11). The amino acid sequences for human lens
A- and
B-
subunits were published previously (12, 13). The Pierce bicinchoninic
acid (BCA) assay was used to determine protein concentrations in the
previous report (10). Protein concentrations determined by extinction
coefficient are considerably greater than those determined by BCA
assay.
Intermolecular Exchange Probed by IEF--
Exchange between A
and
B was studied with a fixed ratio (1:1) (concentration of each, 1 mg/ml) at various incubation times and with various ratios at a fixed
incubation time at 37 °C. IEF was performed on precast
IsoGel-agarose IEF gels (pH range, 3-10; FMC BioProducts, Rockland,
ME) under native conditions. Gels were stained with Coomassie Brilliant
Blue R-250.
CD Measurements--
Protein secondary and tertiary structural
changes were investigated by far- and near-UV CD measurements in an
Aviv circular dichroism spectrometer (model 60 DS) (15, 16). The
reported CD spectra are the average of five 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
dmol1.
Light-scattering Measurement--
Conformational stability was
determined by treatment with heat (17). Aggregation induced by heat was
measured by light scattering in a Shimadz fluorometer (model
RF-5301PC). Briefly, a protein solution of A- or
B-crystallin or
mixtures of various ratios were incubated at 37 °C for 36 h.
Light scattering was measured at 62 °C maintained by a Brinkmann
Lauda circulator (model RCS-6). Preliminary measurements at
temperatures lower and higher than 62 °C were also made, but under
these temperatures aggregation was either too slow or too fast.
Scattering intensity was recorded for 20-60 min with the instrument
set at the same excitation and emission wavelength (400 nm).
FPLC Gel Filtration--
Aggregation or change in quaternary
structure was studied with FPLC gel filtration on a Superose-6 column
(Pharmacia-LKB FPLC system) (14). Protein solutions (A- or
B- or
3-to-1 mixtures; 1 mg/ml) were heated to 62 °C for 20 min and then
cooled to room temperature. The samples were filtered (0.45 µM), and the absorption reading indicated no loss of any
sample due to preincubation. Then the samples were applied to
Superose-6 column, and a fraction of 1 ml was collected in each tube
with an eluting rate of 0.3 ml/min.
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RESULTS |
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Intermolecular Exchange between A- and
B-Crystallins
IEF Study--
IEF shows that there was a
time-dependent exchange between A- and
B-crystallin
subunits at 37 °C (Fig.
1A). The exchange was faster
at 37 °C than at 25 °C (data not shown). The original
A and
B bands became blurred with time, and eventually only one band was
observed; a steady-state was reached after incubation for 30 h.
The focusing pattern did not change upon further incubation. It should
be noted that IEF of
-crystallin in the native state showed a broad
band possibly because of microheterogeneity in the charge or structure.
Similar IEF patterns were observed by van den Oetelaar et
al. (18) in an exchange study using calf lens
A- and
B-crystallins.
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CD Study--
CD of far- and near-UV regions was measured for the
3-to-1 ratio samples of A- and
B-crystallins before and after
exchange. The exchange was induced by incubation at 37 °C for more
than 30 h, which IEF indicated a complete exchange. The lack of
change in far-UV CD (data not shown) but an increase of near-UV CD
(Fig. 2) indicates that intermolecular
exchange causes no gross structural change. An increase of near-UV CD
usually indicates that the protein structure has became more compact.
The controls and
A- or
B-crystallin alone did not show a CD
increase. Near-UV CD of
-crystallin isolated from human infant
lenses was included in Fig. 2 for comparison; it does not overlap
completely with that of 3
A-1
B-crystallin. Apparently, either
in vivo and in vitro intermolecular exchanges are
different or some modifications had already occurred in the human lens
sample.
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Thermal Aggregation of A- and
B-Crystallins
Light-scattering Study--
Fig.
3A shows the scattering
intensity at 62 °C for A- and
B-crystallin and for mixtures of
various ratios of
A to
B with a fixed concentration of
B-crystallin. The samples were incubated at 37 °C for 30 h
to ensure a complete exchange between
A and
B subunits.
B-Crystallin alone displayed an increasing intensity with time, but
the intensity of
A-crystallin alone increased very little,
indicating that
B-crystallin was more susceptible to aggregation.
The addition of
A-crystallin to
B-crystallin suppressed
aggregation, and the extent of suppression increased with increasing
amounts of
A-crystallin until a 3-to-1 ratio was reached. Further
increase in
A-crystallin decreased the suppression; the sample of
3-to-1 ratio was the most stable aggregate. Fig. 3B is a
summary of the scattering measurements with the y axis as
maximal change of slope in the light-scattering curves. These results
may indicate that
A-crystallin stabilizes
B-crystallin from
heat-induced aggregation. The aggregation shown in Fig. 3A may not be in a final state of equilibrium; the scattering intensity may continue to increase if further incubation is undertaken. Our
purpose was to compare the initial responses by these samples, from
which we may infer their thermostability.
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CD Study--
Tertiary structural change was monitored by near-UV
CD; Fig. 4 shows that CD intensity
decreased and became negative in the whole near-UV region for the
3A-1
B hybrid protein at a high temperature (62 °C). Upon
cooling to room temperature, the original intensity was not restored,
and the CD spectrum was almost the same as that at 62 °C. The
irreversible change was also observed previously in calf
-crystallin
samples (17). The decreased near-UV CD may reflect the loosening of
protein structure by partial unfolding. Similar decreased CD was
observed for homo-protein
A-crystallin (data not shown), but
B-crystallin became completely cloudy after incubation under the
same conditions and needed to be filtered before measurement. After the
insoluble fraction was removed, protein concentration was determined
again;
B-crystallin displayed a similar CD decrease (data not
shown).
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FPLC Gel Filtration--
To determine whether increased scattering
intensity of -crystallin at 62 °C is really due to aggregation,
FPLC gel filtration was performed. The samples were preincubated at
62 °C for 20-60 min and then cooled to room temperature before
being applied to the column. Preliminary results of
A and
B of
the present study and our previous study of calf lens
-crystallin
(17) indicate that the extent of FPLC change is dependent on incubation
temperature and time. The data we present here are fixed in both
parameters (preincubation at 62 °C and for 20 min) so a comparison
can be made. Fig. 5 shows that the peak
position for
A-crystallin shifted very little after heating, but the
peak position for
B-crystallin shifted to a higher molecular weight,
and some HMW aggregates were eluted at void volume. The peak shift for
the 3
A-to-1
B mixture was slightly greater than that of
A-crystallin but less than that of
B-crystallin, and no HMW
aggregate was observed at void volume.
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DISCUSSION |
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The intermolecular exchange between A- and
B-crystallin
subunits was demonstrated by IEF in the native state. Similar results have been reported (18), but the
A- and
B-crystallins used were
isolated from bovine lenses under denatured conditions, and they were
not properly reconstituted (19). Furthermore, the significance of the
exchange was not clearly established. Our use of recombinant
A- and
B-crystallins, which are folded in the native form (10), should
yield results more relevant to physiological conditions. Although
exchange occurs in
A/
B samples with any ratios, a 3-to-1 ratio
appears to be most thermostable. Results of heat-induced aggregation
indicate that
A-crystallin is thermally more stable than
B-crystallin, probably because of
B's greater hydrophobicity
(10). Hydrophobic interaction makes an important contribution to
protein folding at low temperatures, but the hydrophobic entropy
decreases as the system becomes enthalpy-driven at high temperatures.
Thus, in vitro or in vivo aggregation between
A- and
B-crystallin (formation of an 600-800-kDa aggregate with a 3-to-1 ratio) may result from a stabilization effect of
A-crystallin on
B-crystallin. The slight shift in the FPLC peak
for
A and the 3-to-1 mixture preincubated at 62 °C (Fig. 5) may
be due to partial unfolding, which is also apparent in near-UV CD
measurements (Fig. 4). This has been reported in the calf lens
-crystallin (17), but the present study indicates that only
recombinant
B-crystallin undergoes heat-induced HMW aggregation. The
notion of stabilization of
B by
A was recently demonstrated by a
study of
A-knockout mice (20). Mice with the targeted disruption of
the
A gene developed lens opacification starting from the nucleus
and showed dense inclusion bodies that contain mostly
B.
The significance of the intermolecular exchange between A- and
B-crystallin is its stabilization of the protein structure. But how
this stabilization is achieved is difficult to speculate, since we do
not know
-crystallin tertiary and quaternary structures;
-crystallin could not be crystallized for x-ray diffraction study. None of the structural models previously proposed, such as the original
or modified three-layer model (21, 22), the micelle-like structure
(23), or the overlapping two-annulus model (24), can explain the
experimental data. It is obvious that the 3-to-1 ratio provides a
balance in charge and hydrophobicity that stabilizes the protein
structure, but a more definite explanation cannot be attained until the
tertiary structure is determined. The near-UV CD clearly indicates that
hybrid aggregate has a more compact structure than either
A- or
B-crystallin homo-oligomers. The difference of near-UV CD spectra
between in vitro formed 3
A-to-1
B hybrid aggregate and
the human infant lens
-crystallin may indicate that they do not have
exactly the same tertiary structure, probably either because of
different folding and aggregation between in vitro and
in vivo conditions or because of modifications in the human
lens
-crystallin and lack of modifications in the recombinant
-crystallin. The difference is most pronounced in the 290-nm bands,
which originate from tryptophan transition, 1Lb
(15, 25). The red shift of the band in the human lens
-crystallin from recombinant
-crystallin (from 292 to 295 nm) indicates that Trp
residues in the human lens
-crystallin are more buried than in the
recombinant
-crystallin (15, 26). It is difficult to tell what
causes this shift, but it is most likely due to a different folded
structure (either tertiary or quaternary structure); the shape of
near-UV CD spectrum of human lens
-crystallin is very different from
that of recombinant
-crystallin.
The protective binding of -crystallin to the partially unfolded
-
and
-crystallins was reported in in vitro experiments (27, 28). Since no subsequent release of the bound substrate was
observed, we should expect plenty of
-
and
-
complexes in
the lens, especially in the aged lens nucleus where
modification-related partial unfolding is extensive. Instead of dimers,
such as
-
and
-
, HMW complexes were observed (29). In human
lenses, the major component of the soluble HMW and insoluble proteins is
-crystallin;
- and
-crystallins are the minor components (6). The chaperone complex formation cannot completely explain this
phenomenon (19). Therefore, the question arises: why does
-crystallin, whose apparent function is to protect other crystallins from aggregation and denaturation, itself become aggregated and insoluble? Since
-crystallin has been shown to be thermally very stable (5), thermal denaturation and aggregation are unlikely to be
responsible for insolubilization. Another possible unfolding mechanism
is post-translational modification. Lens crystallins have a long
half-life and can accumulate a variety of modifications. Although
-crystallin is thermally more stable than
- or
-crystallin, it
is not more stable in the presence of urea (30, 31);
-crystallin is
totally unfolded in 6-7 M urea or 2.5 M
guanidine HCl, but 6 M guanidine HCl is required for
-crystallin to be completely unfolded. Therefore,
-crystallin may
be more susceptible to modification-induced structural perturbation
than
-crystallin, which may lead to HMW aggregation and
insolubilization. It is possible that when
A-crystallin is
extensively modified, it fails to stabilize
B-crystallin. Recently,
we have observed an extensive modification in urea-soluble
-crystallin of human lenses; IEF and reverse-phase FPLC showed a
greater extent of modification in the
A subunit than in the
B
subunit.2
In conclusion, we have demonstrated that lens -crystallin subunits
A and
B undergo intermolecular exchange, which stabilizes protein
conformation. The hybrid aggregate with a 3-to-1 ratio is the most
stable combination and may explain why most mammalian lenses contain
this ratio of
-crystallin subunits.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant EY 05803 and a grant from the Massachusetts Lions Research Fund, Inc.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Center for Ophthalmic
Research, 221 Longwood Ave., Boston, MA 02115. Tel.: 617-278-0559; Fax:
617-278-0556; E-mail: jliang{at}bustoff.bwh.harvard.edu.
1 The abbreviations used are: HMW, high molecular weight; FPLC, fast performance liquid chromatography; IEF, isoelectric focusing.
2 T.-X. Sun and J. J.-N. Liang, unpublished data.
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REFERENCES |
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