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J Biol Chem, Vol. 274, Issue 34, 24137-24141, August 20, 1999
From the Centre for Cellular and Molecular Biology, Hyderabad
500 007, India
A point mutation of a highly conserved arginine
residue in Molecular chaperones facilitate the correct folding of proteins
in vivo and are instrumental in maintaining them in a
properly folded and functional state. The small heat shock protein
(sHSP)1 family is a group of
closely related proteins that are induced during stress. They bind to
aggregation-prone proteins and act as a reservoir for non-native
folding intermediates, which subsequently are refolded by other
chaperones (1). Many diseases are known to result from defective
protein folding (2). However, the mechanism of "chaperoning" has
not yet been completely understood. Interestingly, A point mutation in these two crystallins is known to cause disease in
humans. The conversion of arginine116 to cysteine in To study the possible structural changes caused by the mutation of this
arginine in Cloning of Human Sequencing and Subcloning of Human Site-directed Mutagenesis--
Site-directed mutagenesis was
carried out by the Kunkel method (17) using the Muta-Gene in
vitro mutagenesis kit (Bio-Rad). The Overexpression and Purification of Human Wild-type and Mutant
SDS-PAGE and Western Immunoblot Analysis of the Wild-type and
Mutant Proteins--
Proteins were analyzed on 12% SDS-polyacrylamide
gels (18) under reducing conditions and stained with Coomassie
Brilliant Blue R250 (Sigma). The recombinant proteins were
electrotransferred to a nitrocellulose membrane using a Milliblot
electroblotter apparatus (Millipore) at 0.8 mA/cm2 for
2 h. Primary antibodies for FPLC Gel Permeation Chromatography--
Multimeric sizes of the
wild-type and mutant proteins were evaluated on a Superose-6 HR 10/30
prepacked column (dimensions 10 × 300 mm, bed volume 24 ml;
Amersham Pharmacia Biotech) with reference to high molecular mass
standards (Sigma). Standards used were thyroglobulin (669 kDa),
ferritin (440 kDa), and catalase (232 kDa).
Fluorescence Measurements--
Intrinsic fluorescence spectra
were recorded using a Hitachi F-4010 fluorescence spectrophotometer
with the excitation wavelength of 295 nm. The excitation and emission
band passes were set at 5 and 1.5 nm, respectively.
Circular Dichroism Studies--
Circular dichroism spectra were
recorded using a JASCO J-715 spectropolarimeter. All spectra reported
are the average of 3 accumulations. Far and near UV-CD spectra were
recorded using 0.05 and 1 cm pathlength cuvettes, respectively.
Assay for Protein Aggregation--
Chaperone-like activity of
the wild-type and mutant proteins was studied by the insulin
aggregation assay. The aggregation of insulin (0.2 mg/ml) in 10 mM phosphate buffer, pH 7.4, containing 100 mM
NaCl, was initiated by the addition of 25 µl of 1 M
dithiothreitol (DTT) to 1.2 ml of insulin at 37 °C. The extent of
aggregation was measured as a function of time by monitoring the
scattering at 465 nm in a Hitachi F-4000 fluorescence
spectrophotometer. The extent of protection by the wild-type and mutant
Cloning of Human Site-directed Mutagenesis--
pET21a expression plasmid is also a
phagemid, and hence it was used to make the single-stranded plasmid DNA
having the coding region for both the genes. Site-directed mutagenesis
was used to produce both Expression and Purification of Wild-type and Mutant
Proteins--
Wild-type and mutant Superose-6 Gel Filtration Chromatography--
To investigate the
effect of mutation on the molecular masses of the mutant proteins,
wild-type and mutant Intrinsic Fluorescence and Circular Dichroism Measurements of
Wild-type and Mutant Chaperone-like Activity--
The mutations R116C and R120G
decrease the chaperone-like activity of both the
We thank S. Sujatha for help with
site-directed mutagenesis and K. Rajaraman for help with circular
dichroism studies.
*
This work was supported in part by the Department of
Biotechnology New Delhi, Goverment of India.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. Tel.: 91-40-717 2241;
Fax: 91-40-717 1195; E-mail: mohan@ccmb.ap.nic.in.
2
While this manuscript was under review, two
reports from an annual meeting (33, 34) and a paper (35) appeared that
gave similar results concerning mutations leading to altered aggregate size and decreased chaperone-like activity, with minor differences in
aggregate weight. These minor variations in aggregate weight could be because of the differences in distribution of aggregates and/or the buffer conditions.
The abbreviations used are:
sHSP, small heat
shock protein;
DTT, dithiothreitol;
DRM, desmin-related myopathy;
wt, wild type;
FPLC, fast protein liquid chromatography;
PAGE, polyacrylamide gel electrophoresis.
Structural and Functional Consequences of the Mutation of a
Conserved Arginine Residue in 
A and B Crystallins*
,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
A and B crystallins was shown to cause autosomal
dominant congenital cataract and desmin-related myopathy, respectively,
in humans. To study the structural and functional consequences of this
mutation, human A and B crystallin genes were cloned and the
conserved arginine residue (Arg-116 in A crystallin and Arg-120 in
B crystallin) mutated to Cys and Gly, respectively, by site-directed
mutagenesis. The recombinant wild-type and mutant proteins were
expressed in Escherichia coli and purified. The mutant and
wild-type proteins were characterized by SDS-polyacrylamide gel
electrophoresis, Western immunoblotting, gel permeation chromatography,
fluorescence, and circular dichroism spectroscopy. Biophysical studies
reveal significant differences between the wild-type and mutant
proteins. The chaperone-like activity was studied by analyzing the
ability of the recombinant proteins to prevent dithiothreitol-induced aggregation of insulin. The mutations R116C in A crystallin and R120G in B crystallin reduce the chaperone-like activity of these proteins significantly. Near UV circular dichroism and intrinsic fluorescence spectra indicate a change in tertiary structure of the
mutants. Far UV circular dichroism spectra suggest altered packing of
the secondary structural elements. Gel permeation chromatography reveals polydispersity for both of the mutant proteins. An appreciable increase in the molecular mass of the mutant A crystallin is also
observed. However, the change in oligomer size of the B mutant is
less significant. These results suggest that the conserved arginine of
the -crystallin domain of the small heat shock proteins is essential
for their structural integrity and subsequent in vivo function.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-crystallin, a
lens protein, was shown to prevent the thermal aggregation of proteins
in a manner similar to molecular chaperones (3).
-Crystallin is a multimer of two gene products, A and B, which
have arisen probably because of gene duplication and which share
sequence homology with other members of the small heat shock protein
family (4). Like other sHSPs, they also show chaperone-like activity by
preventing the aggregation of proteins. Using various non-thermal modes
of aggregation, we have shown earlier that -crystallin prevents the
photoaggregation of 
-crystallin (5), the DTT-induced aggregation of
insulin (6), and the refolding-induced aggregation of proteins (7).
These studies demonstrated that a structural perturbation of
-crystallin beyond 30 °C leads to an increase in its
chaperone-like activity (8). The mutations studied in our current
investigation provide an excellent opportunity to probe structural
aspects related to chaperone-like activity of -crystallin and its
enhancement by a structural perturbation. A and B crystallins
were initially thought to be present only in the eye lens, contributing
to its transparency. Recently the non-lenticular expression of B
crystallin (9) has been shown in heart, muscle, and kidney, suggesting
that it might have a diverse function. The expression of A
crystallin outside of the lens is in trace amounts, but it is one of
the important structural proteins in the eye lens. B crystallin
constitutes 3-5% of the soluble protein in the cardiac tissue (10).
It has been demonstrated to interact with actin, desmin, and other
intermediate filaments (11).
A crystallin
causes congenital cataract (12). The mutation of the corresponding
arginine (position 120) to glycine in B crystallin has been shown to
cause desmin-related myopathy (DRM) as well as cataracts (13). Both of
these inherited diseases are autosomal dominant. DRM results in
weakness of the proximal and distal limb muscles and also involves the
heart tissue, where it causes hypertrophic cardiomyopathy (14). This
arginine is conserved throughout the small heat shock family of
proteins. Modeling studies with the sHSP 16.5 of
Methanococcus janaschii indicate that this
arginine residue (position 107) is buried in the hydrophobic core of
the protein and forms a salt bridge with glycine 41 (15). It has been
speculated that this arginine residue in A and B crystallins has
a role in either substrate binding or protein structure stabilization
thereby influencing the chaperone-like activity. From the involvement
of the conserved arginine in inherited human diseases, it is clear that
it has an important role in the structure and function of sHSPs.
A and B crystallins and its influence on the
chaperone-like activity, we have mutated Arg-116 in A crystallin to
cysteine and Arg-120 in B crystallin to glycine, by site-directed
mutagenesis. We have studied the secondary, tertiary, and quaternary
structures of the mutant proteins as well as their chaperone-like
activity and compared them with the wild-type proteins.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
A and B Crystallin cDNA--
RNA was
isolated from human fetal lenses using Trizol reagent (Life
Technologies, Inc.). The first-strand cDNA was synthesized from 1.5 µg of total RNA using oligo(dT) primer and Moloney murine leukemia
virus-reverse transcriptase (Stratagene) according to the
manufacturer's guidelines. The cDNA was used to amplify the A
and B crystallin genes by polymerase chain reaction with
gene-specific primers having engineered NdeI and
HindIII sites (16). The amplified products of both A and
B crystallins were cloned into a T-vector pCR2.1 (Invitrogen) to
generate pCR2.1-A and pCR2.1-B plasmids.
A and B
Crystallins--
Sequencing was done with M13 forward and reverse
primers using the Big DyeTM terminator cycle sequencing kit
(Perkin-Elmer) in an Applied Biosystems automated DNA sequencer. The
coding regions of both the A and the B genes were found to be
mutationless. The coding regions of the wild-type (wt) A and B
crystallins were removed from pCR2.1 after digestion with
NdeI and HindIII and ligated in to
NdeI-HindIII linearized expression vector pET21a
(Novagen) to produce pET21a-Awt and pET21a-Bwt, respectively.
A mutant in which
arginine116 is changed to cysteine (R116CA) is made by the
conversion of CGC
TGC at the 346th nucleotide of the A crystallin
gene using a non-coding oligonucleotide, which carries the mutation.
Similarly, the B mutant in which arginine 120 is changed to glycine
(R120GB) was generated by the conversion of AGGGGG at the 358th
nucleotide of the B crystallin gene. The oligonucleotides carrying
the desired mutation, which are complementary to the coding strand,
were synthesized and used in the mutagenesis reaction. The sequences of
the oligonucleotides used to generate mutations in A and B
crystallins, respectively, are as follows: R116CA,
5'-GGCGGTAGCGGCAGTGGAACTCACG-3'; R120GB,
5'-TCCGGTATTTCCCGTGGAACTCCCT-3'.
A and B Crystallins--
The expression plasmids pET21a-Awt,
pET21a-R116CA, pET21a-Bwt, and pET21a-R120GB were transformed
into competent Escherichia coli BL21(DE3) cells. Growth,
induction, and lysis of cells and purification of the recombinant
proteins were done essentially as described by Sun et al.
(16) except that ion exchange chromatography was done using Mono Q
FPLC column (Amersham Pharmacia Biotech) in the final step of
purification instead of a DEAE-Sephacel column.
A and B were raised in rabbit. The secondary antibody used was anti-rabbit IgG alkaline phosphatase conjugate (Roche Molecular Biochemicals). The blot was developed using
p-nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate.
A and B crystallins was studied by incubating insulin with the
required concentrations of the A and B crystallin samples for 10 min at 37 °C. Aggregation was initiated by the addition of 25 µl
of 1 M DTT.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
A and B Crystallin cDNA--
The coding
regions of A and B crystallins amplified by polymerase chain
reaction from human fetal lens first-strand cDNA product were
ligated into the plasmid pCR2.1. Double-stranded sequencing showed that
the coding regions were identical to the reported exon sequences from
earlier work (19, 20). Both the A and B crystallin genes were
subcloned into the expression vector pET21a to generate pET21a-A and
pET21a-B.
A Arg-116Cys (R116CA) and B
Arg-120Gly (R120GA) (see "Experimental Procedures"). The
presence of the mutation and the absence of any other changes in the
coding region of either the A or the B crystallin genes was
confirmed by DNA sequencing.
A and B crystallin proteins
were expressed in E. coli BL21(DE3) using the isopropyl
-D-thiogalactopyranoside-inducible pET21a expression
plasmids. Induction of the plasmids resulted in the expression of the
recombinant proteins with a molecular mass of 20 and 22 kDa (on
SDS-PAGE) for A (wt and mutant) and B (wt and mutant)
crystallins, respectively. Some amount of the expressed mutant proteins
was seen in the insoluble fraction after cell lysis. Only the soluble
fraction was used for purification and further studies. The lysate
containing the expressed wild-type and mutant proteins was fractionated
by ammonium sulfate precipitation (30-60% saturation) and passed
through a Sephacryl-S300 HR gel filtration column, which removed most
of the contaminating E. coli proteins. Following gel
permeation chromatography, the proteins were purified to homogeneity by
passing through a Mono Q FPLC ion exchange column. Fig.
1 shows the SDS-PAGE patterns for
wild-type and mutant proteins. The recombinant proteins were purified
to ~95% homogeneity as evident from the Coomassie Blue-stained
SDS-PAGE gel. Western immunoblotting using anti-human A and B
crystallin antisera confirmed their expression.
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Fig. 1.
SDS-PAGE of wild-type and mutant
A and B crystallins.
Lane 1, low molecular weight protein standards; lane
2, purified A wild-type crystallin; lane 3, purified
R116CA mutant; lane 4, purified B wild-type
crystallin; lane 5, purified R120GB mutant.
A and B crystallins were chromatographed on
a FPLC Superose-6 gel filtration column. The aggregate sizes of the
individual homo-oligomers of wild-type A and B crystallins were
observed to be slightly smaller than reported for the -crystallin
heteroaggregate. This finding is consistent with earlier published
reports (16, 21, 22). Electron microscopy has revealed that the
recombinant -crystallin complexes are characterized by a
polydisperse morphology (23). The sizes of the complexes depend on the
physicochemical conditions (24). In the present study, the average
molecular masses of wild-type A and B crystallins were observed
to be ~640 and ~620 kDa, respectively. The arginine mutation
altered the oligomer size in R116CA and R120GB (Fig.
2). The R116CA homoaggregate was
observed to be highly polydisperse in nature. The experiment was
repeated several times and at different concentrations, confirming the
polydispersity. Berengian et al. (25) also observed a
significant change in the elution volume for the R116CA mutant. They
have suggested that this change may occur either because of an increase in the number of subunits in the oligomer or because of structural rearrangement in the subunits. We have observed that R116CA forms a
wide range of large aggregates, with larger aggregates exceeding 2000 kDa, as shown in Fig. 2A. It is possible that this large increase in size is due to the increase in the number of subunits in
the oligomer. The elution pattern of R120GB protein is shown in Fig.
2B. The R120GB mutant shows an apparent molecular mass of
~720 kDa. The profile suggests the presence of species larger than
the wild-type B crystallin. We have also observed some species having lower molecular masses than the wild-type protein.
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Fig. 2.
FPLC gel filtration profiles of wild-type and
mutant A and B
crystallins on a Superose-6 column. A, Awt
crystallin (
) and R116CA (····). B, Bwt
crystallin () and R120GB (- - -). The void volume
(a) and elution positions of thyroglobulin (669 kDa)
(b), ferritin (440 kDa) (c), and catalase (232 kDa) (d) are also indicated.
A and B Crystallins--
Intrinsic
fluorescence spectra (Fig. 3,
A and B) show that the emission maxima of the
R116CA and R120GB crystallins remain unaltered (337 nm) relative
to the wild-type A and B crystallins. This finding indicates that
the mutations did not lead to an alteration in the solvent
accessibility of tryptophan residues. They remain relatively
inaccessible to solvent in both the wild-type and the mutant proteins.
However, there is a significant decrease in fluorescence intensity,
suggesting that the tryptophan residue is in a more flexible
environment. Near UV-CD spectra of the R116CA mutant show
significant loss of resolution and change in the 275-295 nm region of
the spectrum (Fig. 4A). The
loss of resolution is probably due to scattering caused by the larger
oligomer size. The changes in the near UV-CD spectra are observed in
the region contributed by tryptophan residues. The near UV-CD spectrum
of the R120GB mutant also shows an alteration in the 270-290 nm region (Fig. 4B). Taken together the results of the
fluorescence and near UV-CD spectroscopy suggest increased mobility of
tryptophan residues and significant changes in tertiary structures of
the mutant proteins. Far UV-CD spectra of wild-type and mutant proteins are shown in Fig. 5, A and
B, respectively. The spectra of R116CA and R120GB
showed distinct changes in the region around 208 nm. Such alterations
in this spectral region were observed by Wynn and Richards (26) in core
mutants of thioredoxin and by Mchaourab et al. (27) in T4
lysozyme. They attributed these changes to an alteration in the packing
of secondary structural elements. An interesting observation is the
change in the cross-over position of the spectra from 204 nm for Awt
to 207 nm for R116CA (Fig. 5A).
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Fig. 3.
Intrinsic fluorescence spectra of wild-type
and mutant A and B
crystallins. A, Awt crystallin () and R116CA
(····). B, Bwt crystallin () and R120GB
(····).
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Fig. 4.
Near UV-CD spectra of wild-type and
mutant A crystallins. A,
Awt crystallin (), R116CA (····). B,
Bwt crystallin () and R120GB (····). The samples were
prepared in 50 mM Tris-HCl buffer, pH 7.4, containing 100 mM NaCl and 1 mM EDTA.
View larger version (21K):
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Fig. 5.
Far UV-CD spectra of wild-type and
mutant A crystallins. A, Awt
crystallin () and R116CA (····). B, Bwt
crystallin () and R120GB (····). The samples were
prepared in 50 mM Tris-HCl buffer, pH 7.4, containing 100 mM NaCl and 1 mM EDTA.
A and B
crystallins, respectively. A 1:1 (w/w) ratio of chaperone to insulin
prevents DTT-induced aggregation of insulin completely when wild-type
A and B crystallins are used. On the other hand, much reduced
chaperone-like activity is observed in the presence of mutant proteins.
Similar ratios show decreases in the protective ability to different
extents for the two mutants. In the case of R116CA, a 1:1 (w/w)
ratio shows only 15% protection. Increasing the chaperone
concentration to 1.5 and 2 times the insulin concentration does not
give complete protection. As shown in Fig.
6A, a 1.5:1 ratio gives only
60% protection from aggregation, and a 2:1 ratio of chaperone to
insulin shows 75% protection. The initial scatter value for the
R116CA alone without insulin was very high. The large initial
scatter value suggests a large molecular size of the R116CA mutant.
The data were normalized to determine the protective effect of the mutant protein. The protection ability of R120GB also decreased compared with the wild-type protein (Fig. 6B). At a 1:1
ratio of the mutant protein to insulin, the observed protection is
40%, whereas the wild-type protein exhibits complete protection.
Increasing the R120GB concentration 2-fold shows only 70%
protection. These results suggest that the arginine mutation impaired
the protective ability of both A and B crystallins. They also
indicate that this arginine residue has an important role in
maintaining the structure and hence the chaperone-like activity of A
and B crystallins. It is interesting to note that this arginine
residue is conserved in 28 species of mammals and other vertebrates
such as chicken and frog (28). In an earlier site-directed spin
labeling study, Arg-116 was shown to be present in a -strand located
near a subunit interface forming a buried salt bridge (25). Other
site-directed mutation studies with both A and B crystallins
resulted in the loss of chaperone activity whenever charged residues
were mutated (29, 30). These studies demonstrate the need for
-crystallins to conserve their net charge through evolution (28).
The present study also shows that disturbing the net charge of the
-crystallins alters the structure and reduces the chaperone-like
activity. The -crystallin domain in particular is highly invariant
across different species signifying its functional importance.
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Fig. 6.
Chaperone-like activity of wild-type and
mutant A and B
crystallins. A, Effect of A crystallin. DTT induced
aggregation of 0.2 mg/ml insulin alone (a) and in the
presence of 0.2 mg/ml (1:1 w/w) Awt (b). Curves
c, d, and e represent the aggregation of
insulin at ratios of 1:1, 1.5:1, and 2:1 R116CA:insulin,
respectively. B, effect of B crystallin. DTT induced
aggregation of 0.2 mg/ml insulin alone (a) and in the
presence of 0.2 mg/ml (1:1 w/w) Bwt (b). Curves
c, d, and e represent the aggregation of
insulin at ratios of 1:1, 1.5:1, and 2:1 R120GB:insulin,
respectively.
B crystallin is known to interact with intermediate filaments under
stress (31). It exhibits a weak interaction with desmin in
physiological conditions. Under conditions of ischemic stress where
there is acidification in the cytosol, the binding affinity of B
crystallin to desmin increases considerably (11). In DRM, aggregates of
desmin and mutant B crystallin could be seen in patient muscle
biopsies. From the protein aggregation assay, it is clear that
R120GB has reduced chaperone-like activity when compared with the
wild-type protein.
A crystallin is present mainly in the eye lens and is found in
traces in non-lenticular tissues. The mutation R116C in A crystallin
causes congenital cataracts. Because B crystallin is present in the
eye lens as well as other tissues such as heart, kidney, and muscle,
the mutation R120G is involved in causing DRM along with cataract. Even
though A and B crystallins have 55% amino acid identity (32),
the mutation of the conserved arginine residue in the two proteins
shows structural and functional changes in the mutants to different
extents, signifying differences in these two proteins. Further studies
on the mutants should prove useful in understanding the molecular
mechanism of the chaperone-like activity of -crystallin and in
designing strategies to prevent or postpone several complications where
-crystallins appear to play a
role.2
ACKNOWLEDGEMENTS
FOOTNOTES
Recipient of a Junior Research Fellowship from the University
Grants Commission, Government of India.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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