Preferential Interaction of Alpha Crystallin With
Denatured Forms of Gamma Crystallin
5. Gopalakrishnan, D. Boyle, and L. Takemoto
Purpose. To characterize the possible interaction of alpha crystallin with partially denatured
forms of gamma crystallin.
Methods. Gamma crystallin was denatured in the presence of guanidine hydrochloride, then
dialy/ed in the presence or absence of alpha crystallin. The high-molecular-weight complex
formed in the presence of alpha was characterized by gel filtration chromatography, electron
microscopy, and quantitative Western blot analysis.
Results. Relative to native alpha or reconstituted aggregates of purified alpha, the higher
molecular weight complex possessed a greater mean diameter and contained increased
amounts of gamma crystallin.
Conclusions. Alpha crystallin preferentially interacts with partially denatured forms of a lens
protein, consistent with its putative role as a functional molecular chaperone in the intact lens.
Invest Ophthalmol Vis Sci. 1994;35:382-387.
1 he alpha crystallins are one of the most abundant
classes of proteins in the lens. They comprise the alpha-A and alpha-B chains, that exhibit extensive homology in their sequences. 1 Besides acting as a medium for the refraction of light, recent studies have
demonstrated that the alpha crystallins can protect
other polypeptides against the denaturing effects of
heat-induced aggregation. 23 These results are consistent with sequence homologies between the alpha
crystallins and previously characterized heat shock
proteins, 45 which are expressed in increased amounts
during heat-induced stress of nucleated cells.
The molecular chaperones consist of highly conserved groups of polypeptides that serve a variety of
roles in the cell. They preferentially bind to partially
denatured forms of polypeptides, protecting them
against further denaturation or facilitating protein
renaturation or both. They can also act as "chaperones" by binding to partially denatured proteins and
facilitating their translocation to other parts of the cell
(see references 6 and 7 for a review of these functions).
From the Division of liiology. Kansas State University, Manhattan, Kansas.
This research was supported by grants from the Nil! and NASA.
Submitted for publication: May 13. 1993: revised July 16, 1993; accepted August
9, 1993.
Proprietary interest category: N.
Reprint requests: L. Takemoto, Division of liiology. Kansas State University, Ackert
Hall, Manhattan, KS66506--I901.
382
Because the alpha crystallins can protect other
proteins against heat-induced aggregation using an in
vitro assay, and because the alpha crystallins are related in sequence to heat shock proteins, it is highly
possible that one of the major functions of the alpha
crystallins in vivo is to bind to and prevent further
denaturation of lens proteins that have been subjected
to both physical and chemical forms of stress found in
the intact lens. In this report, we demonstrate that the
alpha crystallins do indeed interact directly and preferentially with denatured forms of the lens gamma crystallins.
MATERIALS AND METHODS
Newborn bovine lenses were obtained from Antech,
Inc. (Tyler, TX), and were stored at —75°C until use.
In the authors' opinion, methods for obtaining tissue
adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Alpha and
gamma crystallins were purified from the water-soluble fraction of the lens cortex as described previously,8
using a TSK G3000SW column (Perkin-Elmer, Norwalk, CT). The amount of protein in each fraction was
determined according to the BCA method (Pierce
Chemical Co., Rockford, 1L), using bovine serum albumin as standard. After dialysis and lyophilization,
the gamma crystallin fraction was reduced and carboxInvestigative Ophthalmology & Visual Science. 1-Vbn.iary 1094, Vol. 3"). No. 2
Copyright © Association for Research in Vision and Ophthalmology
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Alpha-Gamma Interaction
yamidated (RCA) in the presence of 7 M guanidine
hydrochloride,9 followed by dialysis and lyophilization. To determine the possible interaction of alpha
with different forms of gamma crystallin, 1.80 mg alpha was mixed with 0.36 mg native or RCA gamma in
0.50 ml solution containing 6.0 M guanidine hydrochloride, 10 mM Tris-HCl, pH 7.4. After incubation at
22°C for 15 minutes, the solution was dialyzed for 30
to 36 hours at 4°C against a solution containing 10
mM Tris-HCl, pH 7.4, with several changes of the
buffer, then dialyzed for 4 to 6 hours against TSK
buffer (0.06 M sodium phosphate, 0.1 M sodium sulfate, pH 7.0). The dialysate was centrifuged at 10,000g
for 5 minutes, and 50% of the supernatant was injected into a Biosep S4000 gel permeation column
(300 mm X 7.8 mm, Phenomenex, Torrance, CA).
Proteins were resolved at a How rate of 0.5 ml/min,
using TSK buffer.
The major peaks were collected, and 5% of the
material was precipitated in acetone to remove salts,
then dissolved in sample buffer and analyzed by Western blot analysis using 15% (v/v) polyacrylamide gel
electrophoresis.10 For the purposes of quantitation,
known amounts of alpha crystallins, gamma crystallins,
or RCA gamma crystallins were resolved at the same
time. The blots were probed with rabbit polyclonal
antisera made against human alpha and bovine gamma
crystallins, which were furnished by Dr. S. Zigler, National Eye Institute. After binding of radioiodinated
protein A, protein bands of the resulting autoradiograph were quantitated using scanning densitometry.
Approximately 10% of the remaining material
from the S-4000 column was applied to Formvar- and
carbon-coated copper grids, stained with uranyl acetate, exposed to osmium tetroxide vapor, and visualized by electron microscopy as previously described.^1) Statistical analysis of aggregate diameters
was carried out according to the Mann-Whitney test,
using the Number Cruncher Statistical Package (J.L.
Hintze, Kaysville, UT).
RESULTS
Figure .1 shows the profiles of various mixtures of alpha and gamma crystallins, after their dialysis and resolution using a Biosep S4000 gel permeation column.
Figure 1A shows the elution times for a mixture of
alpha and gamma crystallins that was not subjected to
prior denaturation with guanidine hydrochloride. The
15.6-minute and 21.6-minute elution times represent
the peaks for native alpha and native gamma crystallins, respectively. Guanidine hydrochloride treatment
and dialysis of alpha alone (Fig. IB), followed by
S-4000 chromatography results in a peak eluting at a
similar time (15.4 minutes).
Denaturation of the binding protein by guanidine
hydrochloride, followed by dialysis or dilution to rena-
383
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RETENTION TIME (MINUTES)
FIGURE 1. Gel permeation chromatography of alpha plus
gamma crystallin, with and without guanidine hydrochloride
treatment.. See Materials and Methods for details of renaturaiion and chromatography. (A) Alpha plus gamma, no guanidine hydrochloride treatment, the vertical arrow designates
the void volume of the column; (B) alpha alone, guanidine
hydrochloride treatment plus dialysis; (C) alpha plus gamma,
guanidine hydrochloride treatment plus dialysis; (D) alpha
plus RCA gamma, guanidine hydrochloride treatment plus
dialysis; (E) gamma alone, guanidine hydrochloride treatment plus dialysis, the vertical arrow designates the expected
elution time of the HMVVA.
lure, is a commonly used procedure to study their potential interactions.12 Under these conditions, the
chaperone binds to proteins in various stages of rena-
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384
Investigative Ophthalmology & Visual Science, February 1994, Vol. 35, No. 2
turation. In Figure JC, alpha crysiallins and unmodified gamma crystallins were incubated in the presence
of 6.0 M guanidine hydrochloride, followed by dialysis
and S-4000 column chromatography. Compared with
the 15.6-minute peak for native alpha in Figure 1A,
guanidine hydrochloride treatment results in an earlier eluting peak (13.5 minutes), termed the high-molecular-weight aggregate (HMWA) peak. Also present
is a smaller peak eluting at 15.5 minutes, which probably represents uncomplexed alpha, and is termed the
native alpha peak.
In Figure ID, the gamma preparation was first
reduced and carboxyamidated before dialysis, to ensure that complete renaturation was blocked. Under
these conditions, the HMWA eluting at 13.6 minutes
was also present when the dialysate was resolved by
S-4000 chromatography.
Figure IE shows that guanidine hydrochloride
treatment of gamma alone, followed by dialysis, does
not result in the HMA peak. The absence of a detectable peak represents the probable precipitation of
gamma. Identical results were obtained for RCA
gamma (results not shown). The lack of a gamma or
HMWA peak in Figure IE reflects the absence of alpha, which binds to gamma and prevents precipitation
during the dialysis renaturation procedure.
Together, the results shown in Figure 1 suggest
that after denaturation and partial renaturation in the
presence of alpha, some of the gamma may bind to
alpha crystallins, preventing its precipitation, and producing an alpha-gamma aggregate of higher molecular weight.
To verify the presence of these higher molecular
weight aggregates, electron microscopy was used to
compare their size with aggregates from native alpha.
Negative staining (Fig. 2) shows the presence of particles that could be measured and quantitated as shown
in the histogram in Figure 3. Relative to native alpha
or reconstituted alpha alone (Fig. 3, A and B), guanidine hydrochloride treatment and dialysis of alpha in
the presence of gamma (Fig- 3C) or RCA gamma (Fig.
3D) produced aggregates with a greater range of size,
resulting in a larger mean value (12.01 ± 3.66 in Fig.
3C; 11.98 ± 3.51 in Fig. 3D) as compared with 9.53 ±
2.31 in Figure 3A and 10.6 ± 3.07 in Figure 3B. Statistical analysis using the Mann-Whitney test demonstrated that the populations of aggregates shown in
Figure 3C and Figure 3D were significantly larger than
those shown in Figure 3B (P <z 0.009).
To characterize the protein composition of the
HMWA plus native alpha peaks versus the peaks from
native or reconstituted alpha, the peaks from S-4000
chromatography were collected, followed by determination of alpha and gamma crystallin by using anlisera
to gamma and alpha crystallin. Figure 4 shows the results of Western blot analysis, which was used to quantitate the amounts of alpha and gamma crystallin as
shown in Table 1. The HMWA plus native alpha peaks
from Figures 1C and ID contain significant amounts
of gamma crystallin (326 ± 24 /zg and 328 ± 12 ^g,
respectively), when compared with the amounts of
gamma found in the major alpha peak obtained from
reconstituted alpha alone (3.4 ±0.3 /ug) or from native
alpha incubated with native gamma (20.4 ± 3.0 fig).
The amount of gamma crystallins present in the
HMWA plus native alpha peaks accounts for approximately 90% of the material added to the original guanidine-hydrochloride-treated mixture, demonstrating
that almost all the gamma has remained in solution by
being complexed with alpha.
DISCUSSION
Recent studies using an in vitro assay have demonstrated that the alpha crystallins are able to protect
other proteins from heat-induced denaturation and
aggregation.2-3 This observation suggests that in the
intact lens, one of the major functions of alpha crysiallins is to protect lens polypeptides against the extensive amounts of denaturation that could eventually result in cataractogenesis. Based on studies of molecular
chaperones from other cell types,13 the mechanism of
this protection must involve direct interaction of alpha
with the partially denatured lens protein.
To test this hypothesis, we characterized the binding of alpha crystallin to native versus denatured
forms of the gamma crystallin. We dissolved both alpha crystallin and gamma crystallin in 6.0 M guanidine
hydrochloride, then renatured them by dialysis. The
results in Figures 1 to 3 clearly show that guanidine
hydrochloride treatment and subsequent dialysis of a
mixture of alpha and gamma crystallin results in the
production of larger aggregates than those obtained
after identical treatment of alpha alone.
The results suggest that, formation of the high-molecular-weight aggregates is due to the preferential
binding of alpha to denatured forms of gamma crystallin, to produce a supramolecular complex containing
both alpha and partially denatured forms of gamma
crystallin. This conclusion is supported by the results
of Table 1, which demonstrate that the high-molecular-weight aggregates contain much larger amounts of
gamma or RCA gamma than do the aggregates of native alpha or alpha reconstituted in the absence of
gamma. Using identical conditions to denature and
renature gamma crystallin in the presence of alpha, we
recently showed that gamma preferentially binds to
the central region of the alpha aggregate.14 Because
binding to the central region of the aggregate has been
hypothesized to be a common characteristic of molecular chaperones,1516 these results suggest that the alpha-gamma interaction is not due to nonspecific aggregation but rather is the result of a specific interaction between partially denatured forms of gamma and
the chaperone-binding site of alpha crystallin.
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Alpha-Gamma Interaction
385
1
FIGURE 2, Electron microscopy of aggregate peaks from S-4000 chromatography. See Materials and Methods for details of electron microscopy. (A) Alpha crystallin peak from alpha
plus gamma, no guanidine hydrochioride treatment; (B) alpha alone, guanidine hydrochlortde treatment plus dialysis; (C) HMWA plus native alpha peaks, alpha plus gamma, guanidine hydrochioride treatment plus dialysis; (D) HMWA plus native alpha peaks, alpha plus
RCA gamma, guanidine hydrochioride treatment plus dialysis. The inserted bar in panel (D)
represents the distance of 50 nm.
Because the gamma crystallins contain an unusually large number of cysteine and half-cystine residues, 1 reduced alkylation of these amino acids with
iodoacetamide would be expected to result in forms of
gamma that would not completely renature under any
condition. The observation that alpha binds to the
same amounts of unalkylated versus RCA gamma after
guanidine hydrochioride treatment supports the conclusion that alpha is indeed preferentially recognizing
denatured forms of the gamma crystallin structure.
This conclusion is consistent with the results of another recent study, which showed that alpha can inter-
act directly with the enzyme carbonic anhydrase after
its denaturation by heat. 17
It should be realized, however, that the amount of
gamma crystallin that binds alpha crystallin after
guanidine hydrochioride denaturation and dialysis will
depend on several parameters. These include the
weight ratio of alpha to gamma, the concentration of
the denaturant, and the identity of the protein being
denatured. For example, it was previously shown that
alpha crystallin facilitated the renaturation of gammas
crystallin to a conformation similar to that of native
protein. 18 Consistent with these earlier findings, we
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Investigative Ophthalmology & Visual Science, February 1994, Vol. 35, No. 2
SSi
B
1 2
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I
I
D.
20
X =11.98 +3.51 nm
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5
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2 3
4
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FIGURE 4. Western blot analysis of major peaks obtained
from S-4000 chromatography. Approximately 2% (A) or 1%
(B) of the collected peaks were analyzed as described in Materials and Methods, using polyclonal antisera made against
gamma crystallin (A) or alpha crystallin (B). Arrows designate the bands quantitated by densitometry. Lane 1 of (A)
represents 2.5 Mg of gamma crystallin standard, and lane 1
of (B) represents 2.5 /ig of alpha crystallin standard. Lanes 2
to 5 of (A) and (B) represent the same samples. Lane 2,
native alpha peak from alpha plus gamma, no guanidine
treatment; lane 3, native alpha peak from alpha alone, guanidine treatment plus dialysis; lane 4, HMWA plus native alpha peak, alpha plus gamma, guanidine treatment plus dialysis; lane 5, HMWA plus native alpha peak, alpha plus RCA
gamma, guanidine treatment plus dialysis.
o
I
3 4
25
Diameter (nm)
FIGURE 3. Size distribution of aggregates. The panels correspond with the same samples shown in Figure 2. For each
panel, 100 aggregates were measured, and their relative size
was plotted as a function of percentage abundance. The insert represents the mean ± SD.
lens proteins. Previous studies of other molecular
chaperones have demonstrated that their binding to
denatured forms of proteins can be reversed in the
presence of adenosine triphosphate.67*13 Based on the
results of our in vitro binding studies, most, if not all,
of the binding between alpha and the denatured protein cannot be reversed with the addition of adenosine
triphosphate (results not shown). If a similar situation
exists in the intact lens then the relatively large
amounts of free alpha found in the water-soluble fraction of young lenses may over the lifetime of the organism be irreversibly complexed with partially denatured
proteins produced during the aging process. This pos-
l, Quantitation of Alpha and
Gamma Crystallins in the HMWA and/or
Native Alpha Peaks from S-4000 Gel
Permeation Chromatography
TABLE
have found that after guanidine hydrochloride treatment and dialysis, only about 20% to 30% of gammas
binds to alpha, whereas the rest is eluted in the uncomplexed form (results not shown).
Previous studies have demonstrated the presence
of increased amounts of high-molecular-weight aggregate material in the aging and cataractous lens.19
Some of this material could be the result of alpha binding to partially denatured forms of the other lens crystallins. Based on the results of this report, we hypothesize that during aging, the large amounts of alpha present in lens fiber cells are necessary for the purpose of
binding to and preventing further denaturation of
Sample
Native alpha + native
gamma
Guanidine-treated alpha
Guanidine-treated alpha
+ gamma
Guanidine-treated alpha
+ RCA gamma
Alpha (fig)*
Gamma (fxg)*
1396 ± 46
1446 ± 46
20.4 ± 3.0
3.4 ± 0.3
1456 ± 120
326
± 24
1376 ± 114
328
± 12
* Average of three separate determinations ± SD. Values normalized to starting amounts of alpha and gamma crysiallin.
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Alpha-Gamma Interaction
sibility is consistent with the known, age-dependent
decrease of alpha crystallin in the water-soluble fraction of the lens homogenate. 20 ' 21 As a result of this
binding and insolubilization process, the aged lens
contains much lower amounts of free alpha, resulting
in a diminished ability of the lens to prevent further
denaturation of its proteins.
Key Words
alpha crystallin, molecular chaperone interaction
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