From the Department of Biological Sciences and
§ Department of Chemistry, The University of Wollongong,
Northfields Avenue, Wollongong, New South Wales 2522, Australia and the
¶ Department of Biochemistry, The University of Sydney, Sydney,
New South Wales 2006, Australia
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ABSTRACT |
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Clusterin is a highly conserved protein which is
expressed at increased levels by many cell types in response to a broad
variety of stress conditions. A genuine physiological function for
clusterin has not yet been established. The results presented here
demonstrate for the first time that clusterin has chaperone-like
activity. At physiological concentrations, clusterin potently protected glutathione S-transferase and catalase from heat-induced
precipitation and Clusterin was first described in 1983 as a major secretory
glycoprotein produced by ram Sertoli cells (1). It is a 75-80-kDa disulfide-linked heterodimeric protein with about 30% of the mass of
the molecule comprised of N-linked carbohydrate which is
branched, complex, and rich in sialic acid (2). Clusterin is
transcribed from a single structural gene as a full-length mRNA of
1.6-2.1 kilobases (depending on the length of the poly(A) tail) and
the translated product is internally cleaved to produce the two
subunits prior to secretion from the cell. Clusterin is translated with a typical hydrophobic signal peptide, 21 amino acids in length, which
is proteolytically removed during translocation of the protein to the
endoplasmic reticulum lumen (3). Although clusterin is usually secreted
from cells, it has been reported that treatment of HepG2 and CCL64
cells with transforming growth factor One of the most striking things about clusterin is the breadth of its
biological distribution. In animal tissues clusterin mRNA is near
ubiquitous, with reports describing its occurrence in locales as
diverse as the rat prostate gland, the velvet antler from red deer, and
quail neuroretinal cells. Across this broad species range clusterin
maintains a remarkably high level of sequence homology, comparisons
between mammalian species typically being in the range of 70-80% (3).
The wide distribution and sequence conservation of clusterin suggest
that the protein performs a function of fundamental biological
importance. Furthermore, increased clusterin expression is found in a
variety of disease states and experimental models of pathological
stress (5), including heat shock (6, 7). Therefore, it is reasonable to
suppose that the biological function(s) of clusterin may have immediate
relevance to disease states and stress conditions.
Many binding interactions between clusterin and other biological
molecules have been described. We have shown that clusterin binds to
immunoglobulins (8), lipids (9), heparin (10), glutathione
S-transferase
(GST),12
and the surfaces of pathogenic isolates of Staphylococcus
aureus (11). Others have reported that clusterin also binds to
terminal complement components C7, C8, and C9 (12), apolipoprotein A-I (13), paraoxonase (14), amyloid Sequence analysis predicts that clusterin has three putative
amphipathic We hypothesized that the function of clusterin expressed during
cellular stress, like the small heat shock proteins (sHSPs), may be to
act in a chaperone-like manner and bind to hydrophobic regions of
partly unfolded, stressed proteins, thereby "solubilizing" them and
protecting cells from the cytotoxic consequences of protein precipitation. As a first step toward testing this hypothesis, we
examined the ability of clusterin to protect a variety of proteins from
stress-induced precipitation in vitro. Our results indicate that clusterin has chaperone-like activity and in vitro is a
potent inhibitor of stress-induced protein precipitation. We report
that, like the sHSPs, this protective effect results from the binding of clusterin to partly unfolded proteins to produce a solubilized high
molecular weight (HMW) complex.
Reagents--
Bovine serum albumin (BSA), catalase,
1-chloro-2,4-dinitrobenzene, glutathione (GSH),
H2O2, iodoacetamide, and Protein Precipitation Assays--
Individual solutions of
clusterin (10-200 µg/ml), catalase (200 µg/ml), or GST (200 µg/ml), or mixtures of clusterin with catalase or GST at the same
final concentrations, were prepared in 0.7 ml of 50 mM
sodium phosphate, pH 7.0, and heated at 60 °C. The light scattering
of the solution at 360 nm was measured every 30 s for a total of
25 min in an automated 7-chambered diode array spectrophotometer
(Hewlett-Packard GMBH, Germany). Individual solutions of clusterin
(10-200 µg/ml), Enzyme Assays--
Catalase was prepared at a concentration of
200 µg/ml in 0.1 M sodium phosphate (pH 7.0) with or
without clusterin (100 µg/ml). Mixtures were heated at 37 or 55 °C
for 30 min. 1.0 µl of the catalase/clusterin mixture was incubated
with 1.0 ml of H2O2 substrate solution (0.12%
(v/v) H2O2 in 50 mM
Na2HPO4, pH 7.0) at 37 °C for 5 min before
the reaction was stopped with 150 µl of 4 M NaOH. The
absorbance of H2O2 was measured at 250 nm on a
Spectramax 250 microplate reader (Molecular Devices, Sunnyvale, CA).
Enzyme activity was measured as a decrease in absorbance. GST was
prepared at a concentration of 200 µg/ml in 50 mM
Na2HPO4, pH 7.0, with or without clusterin (100 µg/ml). Mixtures were heated at 37 or 50 °C for 30 min. GST was
then diluted into substrate solution (1 mM GSH, 2 mM 1-chloro-2,4-dinitrobenzene in 0.1 M
phosphate, pH 7.4) to a final concentration of 4.5 µg/ml and
incubated at 37 °C for 5 min before measuring the absorbance at 350 nm. Enzyme activity was measured as an increase in absorbance,
corresponding to the appearance of
1-S-glutathionyl-2,4-dinitrobenzene.
ELISA--
GST and catalase, at 20 µg/ml in PBS (137 mM NaCl, 2.7 mM KCl, 1.5 mM
KH2PO4, 8 mM
Na2HPO4, pH 7.4), were adsorbed onto ELISA trays (Disposable Products, Adelaide, Australia) for 1 h at
37 °C. In some cases, after the coating step, the trays were heated to 60 °C for 30 min to stress the adsorbed proteins. BSA and
Size Exclusion Chromatography--
Individual solutions of
clusterin, GST, catalase, BSA, or Denaturing Gel Electrophoresis--
Proteins collected in the
exclusion volume peaks from size exclusion chromatography were analyzed
using standard methods by electrophoresis on sodium dodecyl
sulfate-polyacrylamide gels (SDS-PAGE). A Hoefer "Tall Mighty
Small" apparatus (Australian Chromatography Co., Sydney) was used at
a constant current of 30 mA. Gels were stained with standard Coomassie
Blue reagent.
Clusterin Protects Proteins from Stress-induced Precipitation but
Does Not Protect Enzymes from Heat-induced Loss of
Function--
Heating GST or catalase at 60 °C produced extensive
protein precipitation within 30 min, shown by an increase in absorbance at 360 nm (Fig. 1, A and
B). Likewise, reduction of BSA or
To investigate whether clusterin is also capable of protecting enzymes
from stress-induced loss of function, we tested the enzyme activity of
GST and catalase before and after exposure to 60 °C for 30 min in
the presence or absence of clusterin. The presence of clusterin (at
sufficient concentration to provide protection against precipitation;
Fig. 1, A and B) had no effect on the loss of
enzyme activity in either case (Fig. 2).
Overall, these results indicate that clusterin has potent heat- and
reduction-stable chaperone-like properties which are capable of
protecting stressed proteins from precipitation but which cannot
protect GST or catalase from heat-induced loss of enzyme activity.
Clusterin Binds Preferentially to Stressed Proteins--
In ELISA,
clusterin did not bind significantly to any of the non-stressed
proteins tested, with the exception of GST (Fig. 3). Binding of clusterin to unstressed
GST had been previously noted.2 Clusterin showed
significantly increased binding to heat-stressed GST (Fig.
3A) and significant binding to DTT-treated BSA and
Clusterin Interacts with Stressed Proteins to Form a HMW
Complex--
Chaperones such as the sHSPs are known to form HMW
complexes with partly unfolded proteins under stress conditions (32, 33). We investigated whether clusterin forms complexes with stressed
proteins by size exclusion chromatography. It is well known that
clusterin aggregates in aqueous solution (3); at physiological pH,
clusterin exists as an equilibrium mixture of monomers, dimers, and
higher aggregation states.2 There was no significant
difference between the elution profiles of untreated versus
heat-treated clusterin (data not shown), indicating that there was no
change in the aggregation state of clusterin in solution in response to
heat. The
In all cases, when clusterin was added to proteins undergoing heat- or
DTT-mediated stress, analysis of the mixtures by size exclusion
chromatography indicated pronounced formation of HMW species (Fig. 4,
A-D, indicated by solid arrows). The HMW species were not detected in analyses of clusterin alone or any of the individual proteins, whether these molecules were stressed or not. Nor
were they detected in mixtures of clusterin and any of the other
proteins in the absence of experimental stress (Fig. 4,
A-D). In each case, SDS-PAGE analysis of the HMW species in the exclusion volume peaks indicated the presence of both clusterin and
the stressed protein under test (Fig. 5).
Thus, these results indicate that clusterin, like the sHSPs, binds to
stressed proteins to form HMW complexes. The only other possible
explanation for these results is that, in each of four different cases,
when clusterin and the "target" protein are present together in
solution, but not when either protein is present alone in solution,
clusterin and the target protein each undergo stress-induced
homo-oligomerization. This latter explanation seems implausible,
particularly in light of the demonstrations that (i) clusterin inhibits
stress-induced protein precipitation (Fig. 1) and (ii) clusterin binds
to stressed proteins in ELISA (Fig. 3). We also analyzed the protein
mixtures produced for size exclusion chromatography by native gel
electrophoresis (at pH 7.0). When mixtures of clusterin and either GST
or BSA were exposed to heat or DTT, respectively, but not in the
absence of experimental stress, these analyses demonstrated the
formation of a new stress-induced species of unique electrophoretic
mobility (data not shown). Similar analyses of catalase or
It was previously shown that clusterin reduced the aggregation of
a synthetic amyloid When tested in ELISA for binding to the native form of the four
proteins tested in this study, clusterin bound only to GST. However,
clusterin bound more strongly to heat-treated than to untreated GST and
also bound significantly to reduced The results discussed above clearly indicate that clusterin binds to
heat-stressed catalase to form a HMW complex. This was further
confirmed by demonstrating by SDS-PAGE analysis that the G7
(anti-clusterin) mAb immunoprecipitates a complex of clusterin and
catalase from heat-stressed mixtures of the two proteins (data not
shown). Yet, clusterin failed to bind to heat-stressed catalase in
ELISA (Fig. 3B). One possible explanation for this apparent discrepancy is that steric constraints imposed by the solid phase adsorption of catalase may have interfered with binding interactions between the two proteins in this assay format. This hypothesis is
consistent with the demonstration that solution phase, heat-stressed catalase inhibited the binding of clusterin to solid phase-adsorbed, heat-stressed GST (Fig. 3B, inset). Presumably, this
resulted from clusterin complexing with catalase in the solution-phase, leading to less clusterin available for interaction with the
solid-phase GST.
No crystal structure is available for clusterin and very limited
experimental data are available concerning the secondary or tertiary
structure of the protein. However, sequence analysis indicates that
clusterin has three putative amphipathic -lactalbumin and bovine serum albumin from
precipitation induced by reduction with dithiothreitol. Enzyme-linked
immunosorbent assay data showed that clusterin bound preferentially to
heat-stressed glutathione S-transferase and to
dithiothreitol-treated bovine serum albumin and
-lactalbumin.
Size exclusion chromatography and SDS-polyacrylamide gel
electrophoresis analyses showed that clusterin formed high molecular
weight complexes (HMW) with all four proteins tested. Small heat shock
proteins (sHSP) also act in this way to prevent protein precipitation
and protect cells from heat and other stresses. The stoichiometric
subunit molar ratios of clusterin:stressed protein during formation of
HMW complexes (which for the four proteins tested ranged from 1.0:1.3
to 1.0:11) is less than the reported ratios for sHSP-mediated formation
of HMW complexes (1.0:1.0 or greater), indicating that clusterin is a
very efficient chaperone. Our results suggest that clusterin may play a
sHSP-like role in cytoprotection.
INTRODUCTION
Top
Abstract
Introduction
References
induces translation of a
truncated form of clusterin (lacking the signal peptide) which remains
intracellular (4).
peptide (15), gp330 (an endocytic
receptor related to the low density lipoprotein receptor (16)), and a
protein secreted by Streptococcus pyogenes (17). Each
description of a new clusterin-binding interaction has led to a
proposal that clusterin is involved with the specific biological function of the binding partner. Consequently, functions proposed for
clusterin are as diverse as its reported binding partners. Thus, it has
been suggested that clusterin acts as a phagocyte recruitment signal
(18), promotes cell aggregation (19), protects cells from complement
attack (20, 21), regulates apoptosis (18, 22), protects cells at
fluid-tissue interfaces from harsh conditions (23), re-models membranes
(24), and transports lipids (25). None of these has been established as
a genuine physiological function.
-helical regions, a type of secondary structure thought
to be important in mediating interactions with hydrophobic molecules
(26, 27). Many of the reported biological ligands of clusterin are
significantly hydrophobic. Therefore, we reasoned that interactions of
these molecules with clusterin may reflect a general propensity of
clusterin to bind to hydrophobic regions of molecules, regardless of
their biological function. In this context, the recent demonstration
that a highly conserved 14-base pair elements, shared by all vertebrate
clusterin proximal promoters, specifically recognizes the heat shock
factor 1 transcription factor is of significant interest. This 14-base
pair element was shown to be capable of mediating heat shock-induced
transcription (28). These authors suggested that this heat-responsive
element provides an explanation for the high sensitivity of clusterin expression to environmental changes and proposed that clusterin may
function as an extracellular heat shock protein (28).
EXPERIMENTAL PROCEDURES
-lactalbumin were
all obtained from Sigma. Dithiothreitol (DTT) was obtained from
Boehringer-Mannheim (Sydney, Australia). All buffer salts were obtained
from Ajax (Sydney, Australia). GST from Schistosoma japonicum was prepared by thrombin cleavage of recombinant Jun leucine zipper-GST fusion protein and purified by GSH-agarose affinity
chromatography as described (29). Clusterin was purified from human
serum by immunoaffinity chromatography as described previously
(8).
-lactalbumin (2.5 mg/ml), or BSA (2.5 mg/ml), or
mixtures of clusterin with
-lactalbumin or BSA at the same final
concentrations, were prepared in 0.3 ml of 50 mM sodium
phosphate containing 0.1 M NaCl and incubated at 37 °C
with or without 20 mM DTT. During this period absorbance readings at 360 nm were acquired every 5 min for a total of 5 h in
a Spectramax 250 plate reader (Molecular Devices, Sunnyvale, CA).
-lactalbumin were applied to ELISA trays in PBS at a concentration
of 1.0 mg/ml, with or without 20 mM DTT, and incubated
overnight at 37 °C. Plates were then blocked with 1% (w/v)
heat-denatured casein prepared in PBS, pH 7.4 (i.e. 1%
heat-denatured casein). Plate-bound DTT-treated proteins were then
incubated with 5 mM iodoacetamide for 1 h at 37 °C,
in order to exclude the possibility of subsequent formation of
disulfide bonds between clusterin and the other proteins. Clusterin, initially at 10 µg/ml, was then serially diluted in binary steps across the ELISA tray, using 1% heat-denatured casein as diluent, and
incubated at 37 °C for 1 h. To minimize nonspecific binding of
clusterin to the ELISA plates, three washes were then performed with
0.1% (v/v) Triton X-100 in PBS. A mixture of G7, 78E, and 41D
anti-clusterin monoclonal antibodies was used to detect bound clusterin, and DNP-9 was used as an isotype control. All antibodies were used in the form of unpurified hybridoma culture supernatants. The
cell line secreting the IgG1
anti-clusterin mAb G7 was a gift from Dr. B. Murphy (St. Vincent's Hospital, Melbourne,
Australia). 78E and 41D, IgG1
anti-clusterin mAbs, and
DNP-9, an IgG1
mAb that binds specifically to the
2,4-dinitrophenyl group, have been described (30, 31). Bound primary
antibodies were detected with sheep anti-mouse Ig-HRP (Silenus, Sydney,
Australia) using o-phenylenediamine dihydrochloride (2.5 mg/ml in 0.05 M citric acid, 0.1 M
Na2HPO4, pH 5.0, containing 0.03% (v/v)
H2O2) as substrate.
-lactalbumin, and mixtures of
clusterin with one of the other proteins (1 mg/ml clusterin with 2 mg/ml GST or catalase; 0.75 mg/ml clusterin with 2.5 mg/ml BSA or
-lactalbumin) were left untreated or treated with heat (60 °C for
30 min; all solutions in 50 mM
Na2HPO4, pH 7.0) or DTT (20 mM at
37 °C for 5 h; all solutions in 50 mM
Na2HPO4, 0.1 M NaCl, pH 7.0) and
then centrifuged (1 min at 10,000 rpm in a benchtop Microfuge) to
remove precipitated protein. 50 µl of each solution was loaded onto a
25 × 1-cm column of Sephacryl 300 (Pharmacia Biotech, Melbourne,
Australia) equilibrated in PBS containing 3 mM azide.
Separations were performed at a flow rate of 0.25 ml/min using a low
pressure liquid chromatography system equipped with a 280-nm flow cell
(Econosystem; Bio-Rad, Sydney, Australia).
RESULTS
-lactalbumin with DTT
resulted in extensive protein precipitation within 4 h (Fig. 1,
C and D). In contrast, clusterin did not
precipitate when heated at 60 °C for 30 min or when treated with DTT
for 5 h (data not shown). When co-incubated with any of the
proteins subjected to heat or DTT-mediated reduction, clusterin
potently inhibited protein precipitation (Fig. 1, A-D).
Since many chaperones (and also clusterin) exist in solution as
aggregates of an ill-defined number of monomers, a convention that has
been adopted when dealing with the interactions between chaperones and
other proteins is to define stoichiometry in relation to the individual
subunits of the chaperone and the protein with which it interacts. For these calculations we have assumed a molecular mass for intact clusterin of 80 kDa and a subunit mass of 40 kDa. An approximate subunit molar ratio (SMR) of clusterin:GST of 1.0:3.2 was the minimum
required to virtually abolish reduction-induced precipitation (Fig.
1A). Corresponding SMRs for the stabilizing effects of
clusterin on the other proteins tested were 1.0:1.3 (catalase), 1.0:2.3 (BSA), and 1.0:11 (
-lactalbumin) (Fig. 1, B, C, and
D, respectively). In contrast, even at corresponding SMRs as
high as 1.4:1.0, a control protein (ovalbumin) had only a small effect
on the stress-induced precipitation of any of the proteins tested (data
not shown).
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Fig. 1.
Absorbance at 360 nm, as a function of time.
A, GST (200 µg/ml) or mixtures of GST (200 µg/ml) and
clusterin (at the concentrations indicated) heated at 60 °C;
B, catalase (200 µg/ml) or mixtures of catalase (200 µg/ml) and clusterin (at the concentrations indicated) heated at
60 °C; C, BSA (750 µg/ml) or mixtures of BSA (750 µg/ml) and clusterin (at the concentrations indicated) treated with
20 mM DTT at 37 °C; (D) -lactalbumin (750 µg/ml) or
mixtures of
-lactalbumin (750 µg/ml) and clusterin (at the
concentrations indicated) treated with 20 mM DTT at
37 °C. Concentrations of clusterin in mixtures:
, 0 µg/ml;
,
25 µg/ml;
, 50 µg/ml;
, 100 µg/ml;
, 250 µg/ml;
, 330 µg/ml;
, 667 µg/ml. The results shown are
representative of three independent experiments.
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Fig. 2.
Enzyme activity of (A) GST
and (B) catalase following incubation at either 37 or
50/55 °C. Solutions containing the specified enzyme ± clusterin were heated for 30 min at the temperature indicated before
measuring enzyme activity. In each case, the table below the
x axis indicates the conditions applied (indicated by an
x). GST activity was measured as an increase in absorbance
at 350 nm following the addition of substrate, representing the
appearance of 1-S-glutathionyl-2,4-dinitrobenzene. Catalase
activity was measured as a decrease in absorbance at 250 nm,
representing the disappearance of H2O2. Further
description of these activity assays is provided under "Experimental
Procedures." The results shown are representative of many independent
experiments. Each histogram represents the mean of three replicate
measurements and the error bars shown correspond to standard
errors (S.E.) of the mean. In some cases, the S.E. are too small to be
visible.
-lactalbumin (Fig. 3, C and D). Thus, our
ELISA results indicate that, comparing untreated versus
stressed GST, BSA, and
-lactalbumin, clusterin binds preferentially
to the stressed form of each protein. In the ELISA configuration
tested, there was minimal interaction of clusterin with either
untreated or heat-stressed catalase bound to the plate (Fig.
3B). However, the binding of clusterin to solid-phase, heat-stressed GST was significantly inhibited by solution-phase heat-stressed catalase (Fig. 3B, inset).
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Fig. 3.
Results of ELISA measuring the binding of
solution-phase clusterin to native ( ) or stressed (
) adsorbed
proteins. Bound clusterin was detected with a mixture of G7, 78E,
and 41D (anti-clusterin) monoclonal antibodies (
,
). DNP-9 (
)
was used as an isotype control for the detection of clusterin binding
to stressed proteins. Where appropriate, GST (A) and
catalase (B) were stressed after adsorption to ELISA wells
by heating the ELISA trays at 60 °C for 30 min. Where appropriate,
BSA (C) and
-lactalbumin (D) were stressed by
reduction during the adsorption stage by incubating solutions of the
proteins in ELISA wells containing 20 mM DTT. Other details
of these assays are as described under "Experimental Procedures."
The inset in B shows the results of an ELISA
demonstrating that under heat-stress conditions, catalase in solution
inhibits the binding of clusterin to solid-phase adsorbed GST. To
perform this experiment, 2.5 µg/ml clusterin was preincubated at
60 °C for 30 min with the indicated concentrations of catalase
before adding to an ELISA plate coated with GST and incubating the
plate at 60 °C for 30 min. Bound clusterin was detected as described
above. The results shown are representative of three independent
experiments. Each data point represents the mean of three replicate
measurements and the error bars shown are S.E. of the mean
in each case. In some cases, the S.E. are too small to be
visible.
and
chains of clusterin are covalently joined by five
disulfide bonds (3). Following treatment with DTT, there was a modest
decrease in the proportion of clusterin oligomers eluting at a position
approaching that of the exclusion limit of the column (compare traces
for clusterin + DTT in Fig. 4,
C and D, with traces for clusterin alone in
A and B). The resolution of the Sephacryl S-300
column used is limited in the range <100 kDa, however, there was no
evidence of the appearance of 40-kDa species which would indicate
physical dissociation of the
and
chains (Fig. 4, C
and D, striped arrows indicate the approximate
position at which a 40-kDa species would be found). Thus, disruption of
the interchain disulfide bonds by DTT resulted in partial dissociation
of large clusterin aggregates to form monomers (80 kDa species) or
lower order clusterin oligomers but did not appear to cause substantial
dissociation of the
and
chains.
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Fig. 4.
Absorbance traces (280 nm) for size exclusion
chromatographic analyses of individual solutions of clusterin, GST,
catalase, BSA, or -lactalbumin, and mixtures
of clusterin with one of the other proteins. Solutions of
individual proteins or mixtures of clusterin with one of the other
proteins (1 mg/ml clusterin with 2 mg/ml GST or catalase; 0.75 mg/ml
clusterin with 2.5 mg/ml BSA or
-lactalbumin) were left untreated or
treated with heat (60 °C for 30 min: all solutions in 50 mM Na2HPO4, pH 7.0) or DTT (20 mM at 37 °C for 5 h: all solutions in 50 mM Na2HPO4, 0.1 M NaCl,
pH 7.0) and then centrifuged (1 min at 10,000 rpm in a benchtop
Microfuge) to remove precipitated protein. 50 µl of each solution was
loaded onto a 25 × 1-cm column of Sephacryl 300 equilibrated in
PBS containing 0.02% (w/v) azide. Separations were performed at a flow
rate of 0.25 ml/min. The filled arrows in A-D
indicate exclusion volume peaks representing HMW species formed during
incubation of clusterin with stressed proteins. The striped
arrows in C and D indicate the approximate
elution position of a 40-kDa species (corresponding to dissociated
/
subunits of clusterin). Chromatography of 50 µl of 20 mM DTT in 50 mM
Na2HPO4, 0.1 M NaCl, pH 7.0, produced a detectable absorbance peak (third trace from the
top in C and D). The results shown are
representative of at least three independent experiments.
-lactalbumin exposed to heat or DTT, respectively, showed, in each
case, stress-induced changes in the electrophoretic mobility of the
target protein only when it was co-incubated with clusterin during
stress. However, in these cases, under the conditions tested, the
putative complexes formed had similar electrophoretic mobilities to
clusterin alone (data not shown). Taken together, our results indicate
that clusterin binds to stressed proteins to form a HMW complex.
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Fig. 5.
Images of Coomassie Blue-stained SDS-PAGE
gels showing analyses of pure proteins and proteins present in
exclusion peaks obtained by size exclusion chromatography of mixtures
of clusterin and stressed proteins (prepared as described in the Fig. 4
legend). Between 1 and 5 µg of protein was loaded into each
lane. A, samples electrophoresed on a 15% SDS-PAGE gel
under reducing conditions: molecular mass standards (lane
1), BSA (lane 2), -lactalbumin (lane 3),
clusterin (lane 4); exclusion peak proteins obtained
by size exclusion chromatography of DTT-reduced mixtures of BSA and
clusterin (lane 5), and
-lactalbumin and clusterin
(lane 6). The solid arrow indicates the position
of the 70-kDa molecular mass standard. B, samples
electrophoresed on a 12% SDS-PAGE gel under nonreducing conditions:
molecular mass standards (lane 1), catalase (lane
2), GST (lane 3), clusterin (lane 4);
exclusion peak proteins obtained by size exclusion chromatography of
heat-treated mixtures of catalase and clusterin (lane 5) and
GST and clusterin (lane 6). The solid arrows
indicate the positions of the 100- and 160-kDa molecular mass
standards. The results shown are representative of two
independent experiments.
DISCUSSION
-peptide in aqueous solution (15). However,
effects of clusterin on the aggregation of stressed proteins have not
been reported before. The results presented here demonstrate for the
first time that clusterin has chaperone-like activity. Clusterin
potently protects GST and catalase from heat-induced precipitation and
-lactalbumin and BSA from precipitation induced by reduction.
Preliminary experiments indicate that clusterin also protects IgG and
ovotransferrin from heat-induced
precipitation.3 Thus,
clusterin has the ability to protect many different proteins from
stress-induced precipitation. However, in the case of two enzymes
tested, GST and catalase, clusterin was unable to protect against
heat-induced loss of activity. The sHSPs, HSP25, and
-crystallin, also protect catalase from thermally induced precipitation but, interestingly,
-crystallin is capable of protecting against enzyme inactivation while HSP25 is not (34). We have not yet determined if
clusterin has any effects on the recovery of enzyme activity following
a heat shock.
-lactalbumin and BSA. Thus, in
the case of GST,
-lactalbumin, and BSA, these results are compatible
with a model in which stress caused partial unfolding of these proteins
to expose binding site(s) for clusterin. For all four proteins
examined, when clusterin was present in solutions of stressed proteins
and these mixtures analyzed by size exclusion chromatography, a HMW
fraction (of mass greater than the exclusion limit of the column,
1.5 × 106 Da) was detected. In each case, SDS-PAGE
analysis of this HMW fraction indicated that it was comprised of both
clusterin and the target protein. It was concluded that clusterin
formed HMW complexes (of mass greater than 1.5 × 106
Da) with each of the stressed proteins tested. This conclusion was
further supported by native gel electrophoretic analysis of mixtures of
clusterin and stressed proteins (data not shown).
-helical regions, secondary
structures thought to be important in interactions with hydrophobic
molecules (26). In addition, sequence analysis predicts a number of
short regions of hydrophobicity outside those defined by the putative
amphipathic
-helical regions (Fig. 6). Since clusterin is known to bind to a variety of native proteins with
hydrophobic domains, it is likely that clusterin exerts its chaperone-like activity by binding to exposed hydrophobic regions of
stressed proteins. As appears likely for clusterin, sHSPs are known to
bind to exposed hydrophobic regions on stressed proteins to form HMW
complexes (35). The formation of these complexes "solubilizes"
stressed proteins, preventing their precipitation. It is also known
that, generally, when acting alone, sHSPs are unable to protect enzymes
from heat-induced loss of function (33, 36). These characteristics
appear to be shared with clusterin.
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Fig. 6.
Predictions based on analysis of the sequence
of human clusterin cDNA. The hydrophobicity index (MacVector
v4.14, Hopp-Woods scale) is plotted as a histogram display. Predicted
regions of amphipathic -helices are represented by striped
rectangles. Proteolytic cleavage sites for the removal of the
signal peptide and internal cleavage to produce the
- and
-chains
are indicated by dotted and solid arrows,
respectively. Asn residues that serve as attachment points for
glycosylation are marked as solid circles.
The low SMRs of interaction between clusterin and the four proteins
tested (ranging from 1.0:1.3 to 1.0:11) indicate that clusterin is a
very efficient chaperone. In comparison, the sHSPs are less efficient
in their interactions with stressed proteins; the available data
suggest that sHSPs bind stressed proteins at a SMR of one or more
subunits of sHSP to one partially folded protein subunit
(i.e. at best, SMR = 1.0:1.0 (37, 38)). Comparing catalase and GST, the catalase subunit (catalase is a homotetramer of
59.7-kDa subunits) is greater in mass than GST (a 25.5-kDa monomer) by
approximately a factor of 2.3. The SMRs corresponding to the minimum
amounts of clusterin required to inhibit heat-induced precipitation of
catalase and GST were 1.0:1.3 and 1.0:3.2, respectively. Thus on a
molar subunit basis, approximately 2.5-fold more clusterin was required
to prevent heat-induced precipitation of catalase versus
GST, a factor that is very similar to the ratio of their subunit masses
(Table I). A similar analysis reveals
that the subunit molecular mass of BSA is approximately 4.9-fold
greater than that of -lactalbumin and, relative to
-lactalbumin,
on a molar basis, BSA requires 4.8 times more clusterin to effect comparable inhibition of reduction-induced precipitation (Table I).
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These data suggest that (i) for heat-stressed proteins, the efficiency
of the chaperone-like action of clusterin is inversely proportional to
the subunit molecular mass of the target protein, and (ii) a similar
relationship applies to proteins stressed by reduction. However, there
is not a simple uniform relationship (for all the proteins tested,
regardless of the type of stress) between mass and the efficiency of
clusterin chaperone-like action. This is apparent by making comparisons
between heat-stressed proteins and those stressed by reduction (see
Table I). This suggests that there may be a fundamental difference in
the mechanism by which clusterin exerts its chaperone-like action under
conditions of stress induced by heat versus reduction.
Despite this, regardless of the nature of the stress, the data suggest
that steric constraints influence the interaction of stressed proteins
with clusterin. When considering proteins stressed by heat, or those
stressed by reduction, in each case clusterin was able to interact with and stabilize a greater number of smaller versus larger
stressed protein molecules. Interestingly, when a similar comparison is made for the interaction of three reduced proteins (-lactalbumin, BSA, and ovotransferrin) with the sHSP,
-crystallin, a similar relationship is found (38).
Multiple lines of evidence suggest that clusterin can act to protect
cells from environmental stresses. We recently reported that
overexpression of clusterin protected murine L929 cells from the
cytotoxicity of tumor necrosis factor (31). It was earlier reported
that inhibition of clusterin synthesis by treatment of LNCaP cells with
an antisense oligonucleotide enhanced the cytotoxicity of tumor
necrosis factor
and that overexpression of clusterin in these cells
protected them from tumor necrosis factor
-mediated death (39).
Overexpression of sHSPs has also been shown to protect cells from the
cytotoxicity of tumor necrosis factor
(40). Furthermore, following
the exposure of U937 cells to UV-B irradiation, in situ
hybridization detected clusterin mRNA in surviving cells but not in
those that had undergone apoptosis (a control mRNA was detected in
both live and dead cells) (41). Similarly, increased clusterin mRNA
expression was not detected in murine olfactory neurons induced
to undergo apoptosis but was detected in surviving glial cells that
surrounded the neurons (42). Therefore, it appears that clusterin
expression is associated with cell survival.
To summarize, clusterin is a "stress-stable," amphipathic molecule
induced in a variety of disease states and instances of cell stress.
In vitro, clusterin potently inhibits stress-induced protein
precipitation and, in vivo, clusterin expression appears to
be associated with cell survival. We speculate that clusterin expression is up-regulated in cells undergoing stress so that clusterin
may bind to partly unfolded proteins within and/or outside cells to
prevent their precipitation. Similar up-regulated expression of, and an
intracellular action by, sHSPs protects cells from stresses and
promotes cell viability (43). The concentration of clusterin shown here
to protect stressed proteins from precipitation is within the range
normally found in some extracellular fluids in mammalian systems
(e.g. 50-370 µg/ml in human serum and 2.1-15.0 mg/ml in
human seminal fluid (44)), raising the possibility that in these
environments clusterin may act as a constitutively expressed
chaperone. On the basis of the available evidence, we propose that the
chaperone-like activity of clusterin may act to protect cells from heat
and other stresses by a mechanism comparable to that of the sHSPs.
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ACKNOWLEDGEMENTS |
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We thank Dr. Robyn Lindner for experimental advice and Dr. Martin Tenniswood for critical review of the manuscript.
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FOOTNOTES |
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* This work was supported in part by Australian National Health and Medical Research Council Project Grants 951074, 951061, and 980497 (to M. R. W. and J. A. C.).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.
Supported by the Clive and Vera Ramaciotti Foundations. To
whom correspondence should be addressed. Tel.: 61-242-214-534; Fax:
61-242-214-135; E-mail: Mark_Wilson{at}uow.edu.au.
2 S. B. Easterbrook-Smith, unpublished results.
3 D. Humphreys and S. Poon, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: GST, glutathione S-transferase; sHSP, small heat shock protein; HMW, high molecular weight; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; SMR, subunit molar ratio; mAb, monoclonal antibody.
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REFERENCES |
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