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INTRODUCTION |
Desmin-related myopathy
(DRM)1 can be caused by
mutations either in the intermediate filament protein desmin, or in the
small heat shock protein 
B-crystallin. Although several mutations in the intermediate filament protein desmin have been linked with DRM (1,
2), so far only a single mutation, R120G, in the small heat shock
protein B-crystallin has been identified (3). A characteristic
disease pathology links the different causes of DRM, which consists of
aggregates of intermediate filaments containing B-crystallin present
in the muscle cells of affected individuals (3, 4).
Other diseases, such as Alexander's disease (5) and drug-induced
hepatitis (6), are also characterized by intermediate filament
aggregates. These aggregates are typically co-associated with
B-crystallin (7) despite the fact that they involve different intermediate filament proteins. This suggests that the association of
B-crystallin with intermediate filament aggregates is independent of
the specific intermediate filament protein but is rather a generic
response to this pathological rearrangement of intermediate filaments.
These data establish a clear link between B-crystallin, intermediate
filaments, and the disease-induced aggregation of intermediate
filaments (8). The mechanism of intermediate filament aggregation and
the role of small heat shock proteins in this process has yet to be addressed.
The recent assessment of sHSP activity in vitro has been
based upon chaperone assays using either heat (9-11) or chemically unfolded substrates (12). These have been extremely useful for studying
the role of ATP (13), post-translational modifications (11, 14-17),
and specific B-crystallin residues (18-22) in the chaperone
activity of sHSPs. These studies mimic the role of sHSPs in stressed
cells, but they do not identify the physiological targets or the role
of sHSPs in unstressed cells.
The sHSPs are expressed in unstressed cells (8, 23-25) and sometimes
at very high levels (26, 27). In unstressed, non-diseased cells of
muscle (23), astrocyte (24, 25), and epithelial and lenticular origins
(27), B-crystallin is found associated with intermediate filaments.
In the eye lens, there is a unique cytoskeletal filament that is a
stable complex of -crystallin, comprising both B- and
A-crystallin, and lens intermediate filaments (28). This is called
the beaded filament. Similar structures can be generated in
vitro under appropriate coassembly conditions (29). These studies
show that the association of sHSPs with intermediate filament networks
is not just a stress-induced event (30), but is a feature of normal
cells, and suggest a general role for sHSPs in intermediate filament biology.
Small HSPs bind to assembly competent intermediate filament proteins.
This was demonstrated in the lens where a soluble complex of
-crystallin and intermediate filament proteins was immunopurified from lens cytosol (27, 31). It was subsequently demonstrated that sHSPs
could inhibit GFAP as well as vimentin assembly in vitro
(27). These data indicated that the association of sHSPs with
intermediate filament proteins is not restricted to the filamentous form, but includes soluble intermediate filament complexes. From these
observations, it is clear that sHSPs have the potential to influence
intermediate filament assembly (29).
Recently, another aspect of the interaction of sHSPs with intermediate
filaments was identified (32). Using a simple viscosity-based assay,
sHSPs were shown to prevent gel formation by intermediate filaments,
presumably by blocking non-covalent filament-filament interactions
(32). These studies suggested a physiologically important link between
intermediate filaments and sHSPs. In a cellular context, this link may
prevent inappropriate interactions between bundled intermediate
filaments (32). Abrogation of this sHSP function could lead to
intermediate filament aggregate formation. This is not only relevant to
DRM but also to other human diseases where this phenotype is a
pathological hallmark such as Alexander's disease.
In this study, the structural and functional properties of the R120G
B-crystallin are compared with those of the wild type protein. Using
several different in vitro assays, the effect of the
mutation upon the chaperone activity of B-crystallin has been
studied. The UVCD, size exclusion chromatography and proteolysis studies all suggest that the mutation has affected the secondary, tertiary, and quaternary structure of B-crystallin. The mutation also causes a significant reduction in the heat-induced denaturation and a reduction the in vitro chaperone activity of the
protein. In order to understand how the B-crystallin mutation could
cause intermediate filament aggregation as seen in DRM, its effect upon the association with intermediate filaments was studied. The data show
that R120G B-crystallin was incapable of preventing
filament-filament interactions that lead to the formation of an
intermediate filament gel in vitro. This was accompanied by
an apparent increase in the binding of R120G B-crystallin to
intermediate filaments. These data suggest that DRM resulting from the
R120G mutation in B-crystallin occurs as a progressive accumulation
of intermediate filament aggregates brought about by the altered
interaction of B-crystallin with intermediate filaments.
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MATERIALS AND METHODS |
Expression Constructs for Recombinant
B-crystallins--
Total RNA was isolated from a sample of human
soleus muscle (RNeasy kit, Qiagen) and converted into cDNA (Life
Technologies, Inc.; Superscript kit). Human B-crystallin cDNA
was amplified from this cDNA using oligonucleotides
5'-AGCCACCATGGACATCGC-3' and 5'-CTATTTCTTGGGGGCTGCG-3' as forward and
reverse primer, respectively. The amplified product was cloned into the
vector pGEM®-T Easy (Promega) and the sequence confirmed
against the GenBankTM data base entry (accession no.
S45630). The R120G mutation was introduced by two-step polymerase chain
reaction using the pGEM-T Easy human B-crystallin wild type vector
as a template in the first amplification reaction. Overlapping,
complementary oligonucleotides were constructed containing the desired
A 
G mutation at nucleotide position 358 as well as a silent A G mutation at nucleotide position 363, 5'-TTCCACGGGAAGTACCGGATCCCAGC-3' and 5'-CCGGTACTTCCCGTGGAACTCCCTGGAGATGAA-3'. Using all four
oligonucleotides the mutated B-crystallin cDNA was created in
two consecutive amplifications, then subcloned. After verification of
the sequences both the wild type and R120G B-crystallin cDNAs
were subcloned into the NdeI and EcoRI sites of
the vector pET23b (Novagen).
Expression of Recombinant B-Crystallins--
Recombinant
human B-crystallins were expressed in BL21plysS(DE3) in the
expression vector pET23b. Recombinant protein expression was induced
using 0.5 mM
isopropyl-1-thio-
-D-galactopyranoside for 4 h once
the cultures had reached an OD600 of 0.6. Harvested bacterial pellets were resuspended in TEN buffer (50 mM
Tris-HCl, pH 8, 1 mM EDTA, 300 mM NaCl, 0.2 mM PMSF, and 0.1% v/v of protease inhibitor mixture
(Sigma)) and lysed by several freeze/thaw cycles. A supernatant
fraction containing the soluble proteins including the recombinant
sHSPs was prepared by centrifuging the lysate at 15,000 rpm in a JA20
rotor at 4 °C for 30 min. The supernatant was then dialyzed against
column buffer.
Preparation of GFAP, Native, and Recombinant
B-crystallins--
GFAP was purified from porcine spinal cord by
axonal flotation as described previously (33). GFAP was then separated
from the other neuronal intermediate filament proteins using DE52
(Whatman) in 8 M urea, 10 mM Tris-HCl, pH 8, 5 mM EDTA, and 25 mM 2-mercaptoethanol. The
proteins were eluted with a 0-200 mM NaCl linear gradient. All column procedures were carried out at room temperature. Recombinant B-crystallin was purified by both non-denaturing (34) and denaturing methods. In the latter, two diethylaminoethyl (DEAE) column steps were
used. The supernatant fraction obtained from the bacteria was loaded
onto the first column comprising TSK-DEAE 650M (Merck Ltd.) in 20 mM Tris-HCl, pH 8.0, 20 mM NaCl, 1 mM MgCl2, 1 mM EDTA, 1 mM DTT, 0.2 mM PMSF and eluted with a 20-250
mM NaCl gradient. The sHSP-enriched fractions were pooled
and dialyzed into buffer containing 6 M urea, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM DTT, 0.2 mM PMSF. This was then loaded onto
a DE52 column equilibrated in the same buffer. Proteins were eluted
with a 0-200 mM NaCl gradient. All column steps were
carried out at 4 °C. Column fractions were checked for homogeneity
by SDS-PAGE. Protein concentrations were determined by the Bradford
protein assay.
Analytical Size Exclusion Chromatography--
Analytical
size-exclusion chromatography was performed on a Biosep-SEC-S4000,
7-µm, 300 × 7.8-mm column using a Hewlett-Packard high
performance liquid chromatograph with the following mobile phase: 0.1 M potassium phosphate, pH 7.0 and 0.2 M NaCl,
at a flow rate of 0.5 ml/min. High molecular weight protein standards (Amersham Pharmacia Biotech) were used to calibrate the column. The
standard deviation of the molecular masses of wild type
B-crystallin/B-crystallin mutants was determined from the peak
width at its half-height in six independent experiments.
Proteolytic, Spectroscopic, and in Vitro Chaperone
Analyses--
Proteolysis of wild type B-crystallin/B-crystallin
mutants was performed as described previously (22, 35). CD spectra were
measured as described previously (36, 37). The presented CD spectra are
the average of 16 scans, smoothed by polynomial curve fitting. The fit
was checked with a statistical test so that the original data was not
over-smoothed. To calculate molar ellipticity, a residue molecular
weight of 115 was assumed. The proteins were dissolved in 20 mM sodium phosphate (pH7.1) and used at concentrations of
0.5 and 1.0 mg/ml for far- and near-UVCD, respectively, as determined
from calculated extinction coefficients based on a protein amino acid
sequence as described previously (38, 39). The pathlength of the cells
was 10 mm for near-UVCD and 1.0 mm for far-UVCD spectroscopy.
Temperature-dependent precipitation of wild type and R120G
B-crystallin was measured in a Beckman DU640 spectrophotometer equipped with a Peltier temperature controlled cuvette holder. The rate
of temperature increase was 0.1 °C/min. Proteins were dialyzed into
20 mM sodium phosphate, pH 7.1, and the concentration adjusted to 0.5 mg/ml prior to the assay. First derivative calculation was used to determine the temperature at which 50% precipitation had occurred.
Temperature-induced aggregation assays using citrate synthase (10) and
alcohol dehydrogenase (11) as target proteins were performed as
described (13, 40).
Intermediate Filament Assembly, Binding, and Viscosity Assays
Involving B-crystallin--
The sedimentation assay as
developed by Nicholl and Quinlan (27) was used to assess the
ability of sHSPs to inhibit intermediate filament assembly. Purified
porcine GFAP was used for these studies. B-crystallins were added to
GFAP in 8 M urea, 20 mM Tris-HCl, pH 8.0, 5 mM EDTA, 25 mM 2-mercaptoethanol prior to the
assembly assay. The final dialysis was against assembly buffer: 10 mM Tris-HCl, pH 7.0, 25 mM 2-mercaptoethanol,
50 mM NaCl. The experiments were performed at room
temperature. Dialysates were layered onto a 0.85 M sucrose
cushion in the assembly buffer and centrifuged at 80,000 × g for 30 min at 20 °C in a Beckman TLA-55 rotor using a
TL100 benchtop ultracentrifuge. The pellet and supernatant
fractions were compared by SDS-PAGE as described previously
(27).
For the intermediate filament binding assay, B-crystallins were
again mixed with GFAP in 8 M urea, 20 mM
Tris-HCl, pH 8.0, 5 mM EDTA, 25 mM
2-mercaptoethanol and then stepwise dialyzed into 10 mM
Tris-HCl, pH 8.0, 25 mM 2-mercaptoethanol. Assembly of the
GFAP intermediate filaments and binding of the B-crystallins was
then induced by addition of a 20-fold concentrated binding buffer (BB)
to give a final concentration of 100 mM imidazole-HCl, pH
6.8, 0.5 mM DTT and incubation at the indicated temperature.
The gel formation assay was based upon a method used to monitor actin
binding protein activity by falling ball viscometry (41). Filament
assembly was promoted exactly as described for the binding assay by
addition of the 20-fold concentrated BB. 100 µl of sample was loaded
into a glass tube to be used in the viscosity assay. This was then
immersed in a 37 °C water bath for 1 h prior to conducting the
gel formation assay. A ball was then placed into the tube, and the
ability of the solution to support the ball was monitored.
Electron Microscopy--
Protein samples were diluted to
100-200 µg/ml, and negatively stained using 1% w/v uranyl acetate.
Grids were examined in a Jeol 1200EX TEM, using an accelerating
voltage of 80 kV.
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RESULTS |
Structure and Stability Characteristics of the R120G
B-crystallin Compared with Wild Type--
The effect of the disease
causing mutation R120G on the structure of B-crystallin was examined
using near- and far-UVCD spectroscopy. The wild type and mutant
B-crystallin were expressed in Escherichia coli using a
pET-based vector system and purified to homogeneity by ion exchange
chromatography. The sample purity was assessed by SDS-PAGE and is
presented in Fig. 1.
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Fig. 1.
SDS-PAGE analysis of purified wild type
B-crystallin and mutant R120G
B-crystallin. A 12% w/v gel was used as the
separating gel and proteins were visualized using the dye, Coomassie
Blue R250. Molecular size standards (lane M;
Mr × 10 3) are shown and labeled
adjacent to lane 1. Lane 1, overexpression of
wild type B-crystallin in E. coli; lane 2, 1 µg of purified wild type B-crystallin; lane 3, 1 µg
of R120G B-crystallin. Position of the B-crystallin is indicated
by an arrow.
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Using recombinantly produced proteins, the far-UVCD spectrum (200-250
nm) was measured for both wild type and mutant B-crystallin (Fig.
2, A and B). At
25 °C (Fig. 2A), the far-UVCD spectrum of wild type
B-crystallin contained a peak minimum at approximately 215 nm that
is consistent with previous UVCD analyses showing a high percentage of
-sheet/-turn structure in B-crystallin (34, 37, 42). When
tested under identical conditions, the R120G mutant displayed a peak
minimum that was shifted to a slightly shorter wavelength and had a
significant (44%) increase in negative molar ellipticity in comparison
to wild type B-crystallin at 220 nm (Fig. 2A). A second
measurement was made for both proteins at 45 °C to assess protein
stability at temperatures utilized in the chaperone assays (Fig.
2B). While the magnitude of the peak minimum (215 nm) for
wild type B-crystallin increased approximately 22% at 45 °C,
there was no increase for the R120G mutant (Fig. 2B).
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Fig. 2.
Far-UV CD spectra of wild type
B-crystallin and mutant R120G
B-crystallin at 25 and 45 °C. The spectra
at 25 (------) and 45 °C (- - -) for wild type B-crystallin
(A) and R120G B-crystallin (B) are shown and
represent an average of 16 scans, smoothed by polynomial curve fitting.
The protein concentrations were 0.5 mg/ml, and the pathlength of the
cell was 1.0 mm. The CD molar ellipticity units are degrees
cm2/dmol.
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At 25 °C, the near UVCD spectrum of wild type B-crystallin
included four positive ellipticity peaks below 290 nm and a large negative peak at approximately 295 nm (Fig.
3A), consistent with past
studies using B-crystallin (34, 37, 42). In stark contrast, peaks
below 290 nm in the near-UVCD of the R120G mutant were shifted
significantly toward negative ellipticity values at 25 °C and only a
small negative peak was observed at 295 nm (Fig. 3A). For
wild type B-crystallin, increasing the temperature from 25 to
45 °C resulted in a large shift in ellipticity from positive to
negative values below 290 nm, but had essentially no effect on the
ellipticity of the 295 nm peak (Fig. 3B). For the R120G
mutant, an increase from 25 to 45 °C resulted in only a modest shift
toward negative ellipticity values below 290 nm, and did not
significantly affect the ellipticity at 295 nm (Fig. 3B).
Both the far- and near-UVCD data indicate that the R120G mutation has
altered the secondary and tertiary structure of the B-crystallin.
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Fig. 3.
Near-UV circular dichroism spectra of wild
type B-crystallin and mutant R120G
B-crystallin at 25 and 45 °C. The spectra
for wild type B-crystallin (A) and R120G B-crystallin
(B) are shown and represent an average of 16 scans, smoothed
by polynomial curve fitting. The protein concentrations were 1.0 mg/ml,
and the pathlength of the cell was 10.0 mm. The CD molar ellipticity
units are degrees cm2/dmol. Spectra for both
B-crystallins were obtained at 25 (------) and 45 °C
(- - -).
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The quaternary structure of R120G B-crystallin was altered, as
assessed by six independent size exclusion chromatography analyses. The
protein complexes formed by the R120G B-crystallin mutant were
larger (Mr = 823,000 ± 131,000) compared
with the wild type (Mr = 633,000 ± 80,000)
and more disperse, as seen by the broader size distribution given by
the standard deviations.
Since the structure of B-crystallin was affected by the R120G
mutation, we decided to assess its temperature stability. Protein aggregation was detected by light scattering (Fig.
4) and showed that both wild type and
R120G B-crystallin did not aggregate up to 55 °C. Above this
temperature, the mutated protein started to aggregate with
precipitation 50% complete at 57.2 °C, whereas the wild type
B-crystallin remained soluble up to 63 °C, showing 50%
precipitation at 64.5 °C. Furthermore, the aggregation of the R120G
B-crystallin occurred over a wider temperature range than that of
the wild type protein (Fig. 4). Thus, the mutation does appear to
affect the stability of the protein, but only at higher (>55 °C),
non-physiological temperatures.
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Fig. 4.
Temperature stability of wild type and
R120G B-crystallin. Aggregation of wild
type (------) and R120G (- - -) B-crystallin was assessed as a
function of temperature from 25 to 85 °C by light scattering at 360 nm. Protein concentration was 0.5 mg/ml in 20 mM sodium
phosphate, pH 7.1. The temperature was increased by 0.1 °C/min.
Temperatures at midpoint of precipitation were calculated to be 64.5 and 57.2 °C for wild type and R120G B-crystallin, respectively.
Note the different slopes of precipitation for the wild type and the
mutant proteins.
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To assess whether the R120G mutation affected the stability of
B-crystallin below 55 °C, the temperature dependence of the ellipticity at 205 and 217 nm was studied by far-UVCD (data not shown).
A gradual conformational transition was shown between 25 °C and
55 °C for the wild type, but not the mutated B-crystallin. This
is consistent with the far-UVCD spectra (Fig. 2, A and
B, cf. spectra at 25 °C versus
45 °C) and indicates that the stability of the mutated protein was
not affected below 55 °C.
The Effect of the R120G Mutation upon the Chymotryptic Digestion of
B-crystallin--
Chymotrypsin has been used to assess the
susceptibility of B-crystallin to proteolysis (22, 35). ATP has been
shown to increase the protection of some of these sites in the
-crystallin domain against chymotryptic digestion (22, 35). These
assays were used to evaluate the effects of the R120G mutation upon the availability of the chymotryptic sites for digestion as another indicator of changes in the structural characteristics of
B-crystallin (Fig. 5). The R120G
mutation did make the chymotryptic sites more accessible compared with
the wild type (Fig. 5), as the R120G mutant was digested faster than
wild type B-crystallin. The pattern of proteolytic products produced
was qualitatively similar but not the same. Protection of the
chymotrypsin sites was afforded upon addition of ATP to both the R120G
and wild type B-crystallin (Fig. 5). Thus, at this level, the
differences in the digestion characteristics of R120G B-crystallin
suggested that there had indeed been some structural changes as a
result of the mutation, but these did not appear to significantly alter
the region(s) of B-crystallin involved in interacting with ATP.
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Fig. 5.
SDS-PAGE analysis of the chymotryptic
digestion of wild type B-crystallin and
R120G B-crystallin in the absence
(NO ATP) and presence of ATP
(PLUS ATP) at 37 °C. Samples were
taken over a 30-min time course. In the absence of ATP, the pattern of
proteolysis for R120G B-crystallin was similar but not identical to
the pattern observed for wild type B-crystallin. Substantial
increase in the susceptibility to proteolysis was noted for R120G
B-crystallin. The pattern of proteolysis obtained for R120G
B-crystallin in the presence of ATP resembled that of wild type
B-crystallin, although again the mutant was more susceptible than
the wild type protein. These data suggest that the mutation certainly
changed accessibility to the chymotryptic cleavage sites in
B-crystallin. Undigested protein is indicated (*). Molecular size
markers are indicated ( ) and correspond to 36.5, 31, 21.5 14.4, 6 and 3.5 kDa.
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In Vitro Chaperone Assays--
Prior to the discovery of the
physiological substrates for B-crystallin, in vitro
assays were developed based upon heat-induced aggregation of proteins
to assess the chaperone activity of B-crystallin. Two were selected
to cover a range of chaperone: substrate concentrations and also a
range of temperatures (10, 11). As shown in Table I, R120G B-crystallin was worse than
the wild type B-crystallin in both chaperone assays. The R120G
mutant was also tested at molar ratios of 2:1 and 10:1
(protein:B-crystallin) using citrate synthase as the assay substrate
and gave similar results to those obtained at a 1:1 ratio (Table I). In
addition, at both 10:1 and 5:1 (protein:B-crystallin) in the alcohol
dehydrogenase-based chaperone assay, similar results to the 20:1 ratio
were obtained (Table I). Therefore, it is reasonable to expect that the
mutation will compromise the ability of B-crystallin to perform its
chaperone role in muscle and lens cells.
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Table I
The chaperone activities of B-crystallin and R120G B-crystallin
compared using the alcohol dehydrogenase and citrate synthase
assays
The R120G B-crystallin was worse in all instances than the wild type
B-crystallin.
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Intermediate Filament Assembly, Binding, and Gel Forming
Assays--
As demonstrated by the discovery of the disease causing
mutation in B-crystallin, intermediate filaments are a physiological target of B-crystallin. Three assays had been developed that assess
the interaction of B-crystallin with intermediate filament proteins
at different levels (32). These are the ability of B-crystallin to
(a) inhibit filament assembly, (b) bind to and co-sediment with intermediate filaments, and (c) prevent
filament-filament interactions as measured by falling ball viscometry
(32). The effect of the mutation R120G upon B-crystallin chaperone
activity was tested using these assays. The intermediate filament
protein, GFAP, was used. GFAP is a physiologically relevant target
because of the formation of Rosenthal fibers, which contain GFAP
filaments coaggregated with B-crystallin in the neurodegenerative
disease Alexander's disease (5). It is also a type III intermediate filament protein closely related to desmin (43) with similar structural
features and mechanisms of assembly.
In Figs. 6 and
7, the effect upon intermediate filament
assembly (Fig. 6) and the ability to bind to intermediate filaments (Fig. 7) was examined. These assays are conducted at different pH and
salt conditions to optimize for the inhibition of assembly and binding
to intermediate filaments, respectively (30, 32). As can be seen in
Fig. 6, R120G B-crystallin was reduced in its ability to inhibit the
assembly of the intermediate filament protein, GFAP. Compared with the
assembly of GFAP in the absence of B-crystallin (Fig. 6,
lanes 1 and 2), the R120G
B-crystallin was still able to partially inhibit GFAP assembly (Fig.
6, lanes 3 and 4). Moreover, a
sizeable proportion of R120G B-crystallin was found in the pellet
fraction (Fig. 6C, lane 4), even at
22 °C, the temperature of this assay. The wild type B-crystallin
remained almost completely soluble under these conditions (Fig.
6B, lane 4). This was the first
indication that R120G B-crystallin bound more avidly to intermediate
filaments than the wild type protein.
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Fig. 6.
Effect of wild type and R120G
B-crystallin on GFAP assembly in
vitro. In the assembly inhibition assay, GFAP was
assembled in vitro (A) in the presence of wild
type B-crystallin (B) and R120G B-crystallin
(C) in a molar ratio of 1:2 at 22 °C. The supernatant
(S) and pellet fractions (P) were analyzed by
SDS-PAGE and stained with Coomassie R250. The positions of GFAP and
B-crystallin are indicated (B and C). Under
these conditions of assembly, most of the GFAP is sedimented
(A, lane 2), with only a small
proportion of the protein remaining in the supernatant (A,
lane 1). Contrast this result to the assembly of
GFAP in the presence of B-crystallin (B), where >50% of
the GFAP remains in the supernatant (B, lane
3). Notice that using these assay conditions, nearly all the
wild type B-crystallin remained in the supernatant fractions in the
absence (B, lane 1) and presence of
GFAP (B, lane 3). In the presence of
R120G B-crystallin (C), >80% of the GFAP is found in
the pellet (P; C, lane 4).
In the presence of R120G B-crystallin, the soluble GFAP remaining in
the supernatant (C, lane 3) is reduced
compared with the coassembly of GFAP with wild type B-crystallin
(B, lane 3) but greater than the
control assembly for GFAP (A, lane 1).
Notice too that the R120G B-crystallin bound to the filaments in the
pellet fraction (C, lane 4) in
complete contrast to wild type B-crystallin (B,
lane 4) under similar experimental conditions. In
the absence of GFAP filaments, most R120G B-crystallin remained
soluble (C, lane 1) with only a very
small proportion sedimenting under these conditions (C,
lane 2).
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Fig. 7.
Binding of wild type and R120G
B-crystallin to GFAP filaments in
vitro. In this assay, the GFAP assembly was conducted
under conditions that promoted B-crystallin binding to intermediate
filaments (32). The binding is a temperature-dependent
process (30, 32), and the three temperatures selected are indicated
above the relevant gel lanes (A-C). The pellet
(P) and supernatant (S) fractions were analyzed
by SDS-PAGE and stained with Coomassie Blue R250. GFAP assembled
efficiently into filaments under these assay conditions at temperatures
of 22, 37, and 44 °C (A). Most of the GFAP is present in
the pellet fractions (A, lanes 2,
4, and 6, labeled P). The small
proportion of GFAP remaining in the supernatant (A,
lanes 1, 3, and 5,
respectively, labeled S) varies with temperature. In the
absence of GFAP, wild type (B, lanes
1, 3, and 5) and R120G B-crystallin
(C, lanes 1, 3 and
5) remained almost entirely soluble (labeled S).
When GFAP is included in the assay, both wild type B-crystallin
(B, cf. lanes 8,
10, and 12) and R120G B-crystallin
(C, cf. lanes 8,
10, and 12) sedimented with the GFAP filaments.
This was temperature-dependent, as an increasing proportion
of wild type B-crystallin and R120G B-crystallin was found in the
pellet fractions (B and C, lanes
8, 10, and 12). Note that in the
presence of GFAP, an appreciable proportion of the R120G
B-crystallin was pelletable even at 22 °C (C,
lane 8). At 44 °C, all the R120G
B-crystallin was present in the pellet fraction (C,
lane 10). The wild type B-crystallin was found
in the pellet fractions at 37 °C (B, lane
10) and 44 °C (B, lane
12), but larger proportion remained in the soluble fractions
at both temperatures (B, lanes 9 and
11, cf. lanes 11 and
12).
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The increased binding of R120G B-crystallin (Fig. 7C) to
GFAP filaments compared with the wild type protein (Fig. 7B)
was a dramatic effect of the mutation. Even at 37 °C, R120G
B-crystallin was found to bind almost completely to the pelletable
GFAP filaments, whereas the wild type B-crystallin was only
partially bound (<20%). This increased binding was consistent at
different molar ratios, from 1:2 to 2:1 (sHSP to GFAP, respectively)
and was apparently unaffected by the presence of 1 mM ATP
(data not shown).
The increased binding of R120G B-crystallin to intermediate
filaments might be expected to increase its ability to prevent filament-filament interactions either indirectly by steric hindrance or
directly by obscuring the filament-filament interaction sites. Using
falling ball viscometry, this hypothesis was tested. The assay was
developed so that GFAP forms a protein gel in the tube in the absence
of B-crystallin. The buffer conditions are the same as those used
for the filament binding assay. This gel is capable of supporting the
ball used in the viscosity assay. Addition of B-crystallin prevents
gel formation and so permits the ball to sink to the bottom of the tube
(32). This is the result of inhibiting non-covalent filament-filament
interactions rather than preventing filament formation as these assay
conditions were similar to those used in the filament binding assay
(Fig. 7), where no loss of sedimentable GFAP was observed (see also
Ref. 32). As expected, after assembly of GFAP in the absence of sHSP in
the control tube, a gel formed preventing the ball from falling. The
presence of wild type B-crystallin in the assay mixture at a 2:1
molar ratio to GFAP prevented the filaments formed from supporting the
ball in the viscometer. In contrast, the mutant R120G B-crystallin
appeared completely ineffective at inhibiting gel formation and thus
the ball was unable to enter the viscometer. Similar results were
obtained for the ratios 1:2 and 2:1, sHSP to GFAP, respectively, and in
the presence of 1 mM ATP (data not shown). Table
II summarizes data obtained from three
separate experiments.
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Table II
Summary of the data collected for the effect of B-crystallin and the
R120G mutant on gel formation by intermediate filaments as monitored by
a falling ball viscometry assay
GFAP can form a protein gel capable of supporting a small ball bearing.
Including B-crystallin in with GFAP prevented gel formation and
allowed the ball to drop to the bottom of the tube. The R120G mutation
abrogated this activity of B-crystallin.
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The data from the viscosity assays have demonstrated that the R120G
mutation abolished the ability of B-crystallin to prevent gel
formation by failing to inhibit filament-filament interactions required
in this process, despite the increased ability of the mutant R120G
B-crystallin to bind to intermediate filaments (Fig. 7).
Visualization of the B-crystallin Intermediate Filament Complex
by Electron Microscopy--
To examine the association of
B-crystallin with the GFAP filaments during the binding/viscosity
assays, samples were examined using negative staining techniques and
electron microscopy (Fig. 8). At
37 °C, limited binding of wild type B-crystallin to GFAP filament
was observed (Fig. 8A, arrows), whereas the R120G
B-crystallin particles were very closely associated with filaments
(Fig. 8B, arrows) with no particles left unbound.
In the absence of GFAP filaments, both wild type (Fig. 8C)
and R120G B-crystallin (Fig. 8D) formed discrete
particles approximately 15-20 nm in diameter (Fig. 8, C and
D, arrows). These observations correlated with the binding assay results (Fig. 7, B and C),
where almost all of the mutant B-crystallin bound the GFAP filaments
compared with a much smaller proportion of the wild type protein. From the electron micrographs, it appears that the increased binding of the
mutant B-crystallin resulted in a very extensive coating of the
intermediate filaments, leading to filament bundling (Fig. 8B).
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Fig. 8.
Visualization of intermediate filaments
assembled in the presence of wild type and R120G
B-crystallin. Samples were taken at the
completion of the viscosity assay and processed using the negative
staining technique for electron microscopy. In the presence of wild
type B-crystallin (A), the intermediate filaments formed
were long and sometimes had B-crystallin particles attached
(arrows). The filaments were generally not clumped in this
preparation. In contrast, filaments co-assembled in the presence of
R120G B-crystallin (B) were clumped (large arrows). Co-aggregation between intermediate filaments
and R120G B-crystallin particles was seen (B,
arrows). In the absence of intermediate filaments, wild type
(C) and R120G B-crystallin (D) both formed
discrete 15-20-nm particles (arrowheads).
Bar = 100 µm.
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DISCUSSION |
The R120G mutation in B-crystallin affects a highly conserved
residue among the whole sHSP family (44, 45). It is important for the
function of A- and B-crystallin as mutating this residue causes
the human diseases, cataract (46) and DRM (3), respectively. Other
mutational studies using the Mycobacterium tuberculosis sHSP, HSP16.3, and the mammalian sHSPs HSP27 and A-crystallin (21)
also show this highly conserved arginine residue is structurally very
important as it is part of a -strand involved in subunit interactions (21). Recently, the crystal structure of the
Methanococcus jannaschii sHSP, HSP16.5, has been described
(47), and this provided the first atomic details of this conserved
arginine residue. From these data, a direct or indirect role for the
disease causing arginine residue in subunit-subunit interactions was
proposed (48). Although a recent study reported on the structural and chaperone-like properties of R120G B-crystallin (49), we initiated our studies to investigate the interaction with intermediate filaments, the physiologically relevant target of B-crystallin given the fact
the mutation causes intermediate filament aggregation in the disease
DRM (3).
Secondary, Tertiary, and Quaternary Structure of B-crystallin Is
Altered by the Mutation R120G--
The far- and near-UVCD data
presented here for the recombinant B-crystallin (Figs. 2 and 3)
correlate well with previously published data (22, 34, 37, 42).
Increasing the temperature from 25 to 45 °C resulted in shifts in
ellipticity values in the far- (50) and near-UV (50, 51) for wild type
B-crystallin that are consistent with a conformational change. The
far- and near-UVCD spectra obtained for the R120G B-crystallin
demonstrated that the mutation affected the secondary and tertiary
structure of the protein. Interestingly, the UVCD spectrum of R120G
B-crystallin at 25 °C appeared similar to that obtained for the
wild type B-crystallin at 45 °C, suggesting that the R120G
B-crystallin already existed in a more open, even partially
unfolded, conformation at 25 °C. In the closely related protein,
A-crystallin, mutation of the equivalent arginine residue (Arg-116)
to a cysteine and subsequent spin label modification also changed the
secondary, tertiary and quaternary structure of the protein (52). The
R120G mutation in B-crystallin modified the protein structure such
that the accessibility of the chymotryptic sites was increased (Fig.
5).
Changes in the quaternary structure were therefore expected and this is
substantiated by the results obtained from size exclusion chromatography. The R120G mutation induced an increase in the average
Mr and a broadening of the
Mr range of B-crystallin. A similar effect
upon the Mr and therefore the quaternary
structure of A-crystallin was also observed (21, 52) when the
equivalent arginine (Arg-116) was changed. In the case of HSP27,
oligomerization of the -crystallin domain (21) was affected by
mutating the equivalent arginine (Arg-140), but here the
Mr decreased. Obviously, while it can be
concluded that this conserved arginine performs a key structural role,
the effect of changing this residue is not necessarily the same for all
the different sHSPs.
The R120G Mutation Affects the Stability of B-crystallin at
Elevated Temperatures--
At elevated temperatures it has been shown
that -crystallin adopts a more disordered structure (53). A
conformational transition with a midpoint at 60-62 °C was observed
by Fourier-transform infrared spectroscopy, differential scanning
calorimetry, and circular dichroism (53). Using the latter method and
monitoring the temperature dependence of ellipticity at 205 and 217 nm,
a gradual transition was observed for wild type, but not R120G
B-crystallin, over the temperature range of 25-55 °C (see also
Fig. 2A). Thus, in contrast to -crystallin (50, 53),
consisting of A- and B-crystallin subunits, B-crystallin alone
does show a gradual conformational transition in this temperature
range. This is not seen for R120G B-crystallin, as expected from
comparing the CD spectra at 25 and 45 °C, which are very similar
(Fig. 2B). At 64.5 °C and 57.2 °C, however, the
heat-induced precipitation of wild type and R120G B-crystallin,
respectively, was 50% complete as measured by light scattering. The
different profile of temperature-induced protein aggregation of R120G
B-crystallin compared with wild type B-crystallin may reflect the
increased polydispersity of the mutant protein complexes, but this
needs further experimentation.
These data do indicate, however, that B-crystallin is first
stabilized by A-crystallin, as -crystallin does not precipitate even at 100 °C (54). Second, these data indicate that the mutation has increased the susceptibility of the protein to temperature-induced unfolding leading to protein precipitation (Fig. 4). Therefore, the
mutation decreases protein stability, but the effects are most readily
seen at non-physiological temperatures. Nevertheless, the more
important question is whether these structural and stability changes
caused by the R120G mutation affect the biological activity of
B-crystallin.
Previous studies made a very important correlation between structural
changes in -crystallin proteins and their chaperone activity (12,
50, 55). Using hydrophobic probes, a transition at 30 °C was
identified. The exposure of these hydrophobic surfaces was correlated
with an increase in the observed chaperone activity of the
-crystallin proteins (12, 50). It has been suggested that the
polydisperse quaternary structure of B-crystallin oligomers (56) is
an important feature of the chaperone activity (12). In other studies
on A-crystallin, however, it has been possible to uncouple the
changes in secondary and quaternary structure from the chaperone
activity (52). Thus, although the R120G mutation causes changes in the
protein structure and stability, it is important to assess the effect
of the mutation upon the chaperone function of B-crystallin.
Effect of R120G Mutation upon B-crystallin
Activity--
In vitro assays (9) have been
developed to study the protein chaperone function of B-crystallin
utilizing the ability of B-crystallin to protect other proteins
against either heat- or chemically induced denaturation
(e.g. Refs. 10 and 11). These proteins do not necessarily
represent physiological targets for the chaperone function of
B-crystallin, but they have allowed this key function to be studied
in vitro. The studies presented here demonstrate that the
chaperone function of B-crystallin is significantly compromised,
although not completely abolished toward these non-physiological
substrates (Table I). In fact, it appeared that R120G-B-crystallin
was a major part of the insoluble pellet with both alcohol
dehydrogenase and citrate synthase as target proteins (data not shown),
as also reported for lactalbumin (49), suggesting that binding does not
absolutely correlate with chaperone activity.
Although these are important assays, they do not address the most
obvious feature of DRM, which is the collapse of the intermediate filament network into characteristic aggregates containing
B-crystallin (3, 4). Several in vitro assays were used to
investigate the effects of B-crystallin on intermediate filaments
(27, 30, 32). The results presented here show some loss in the ability
of R120G B-crystallin to inhibit intermediate filament assembly
(Fig. 6), but the most dramatic changes concerned the interaction of
R120G B-crystallin with assembled intermediate filaments. First, the
mutation caused B-crystallin to bind more avidly to intermediate
filaments (Fig. 7). Second, R120G B-crystallin could no longer
prevent gel formation by intermediate filaments (Table II). The
increase in binding was visualized (Fig. 8) in the negatively stained
samples of the intermediate filament-B-crystallin complexes formed
at 37 °C. In the presence of wild type B-crystallin, limited
binding to the assembled intermediate filaments was observed (Fig.
8A). In stark contrast, the R120G B-crystallin bound
avidly to the assembled intermediate filaments and even appeared to
induce filament clumping (Fig. 8B). Filament lengths were
comparable in both samples and so, as expected from the sedimentation
assay results (Fig. 8; see also Ref. 57), the differences in the
solution viscosity were entirely due to the different B-crystallins.
The data show the mutation in B-crystallin affects all the different aspects of the interaction with intermediate filaments, but the key
aspect would appear to be the change in the activity of B-crystallin with respect to assembled intermediate filaments. The mutation caused
increased binding to intermediate filaments, which appears to actively
encourage filament-filament interactions. We propose that this, coupled
with the loss of the ability of R120G B-crystallin to prevent those
filament-filament interactions seen in the viscosity assay gels, will
lead to intermediate filament aggregation. Our observations are
supported by the disease pathology that is typified by intermediate
filament aggregation coupled with the association of B-crystallin
(3). It is important to realize that both mutations in B-crystallin
and intermediate filament proteins cause such characteristic
pathologies. Our data suggest that a similar change in the association
of sHSPs as caused by intermediate filament mutations will explain
these disease pathologies.
The R120G mutation in B-crystallin obviously compromises
B-crystallin function in vivo, but the disease phenotypes
were not seen at birth, appearing only in early adulthood (3) and affecting only the lenses and muscles of individuals carrying the
mutation, while the other tissues that express B-crystallin had no
phenotype. Several factors could explain these observations. First,
both wild type and R120G B-crystallin will be expressed together
(3). Second, the R120G mutation apparently does not completely abolish
the chaperone activity (Table I). Finally, both the eye lens (58) and
muscle express other sHSPs, sometimes in high concentrations (59, 60),
which could change the sensitivity of the different tissues.
Collectively, these factors might help delay the onset of the disease
and select the eye lens and muscles as those tissues to be affected by
the R120G mutation in B-crystallin.
B-crystallin
Alters Its Protein Structure, Chaperone Activity, and Interaction
with Intermediate Filaments in Vitro*
§,
, and
TOP
ABSTRACT
INTRODUCTION
