Báo cáo Y học: Suppression of apolipoprotein C-II amyloid formation by the extracellular chaperone, clusterin potx

Suppression of apolipoprotein C-II amyloid formation
by the extracellular chaperone, clusterin
Danny M. Hatters
1
, Mark R. Wilson
2
, Simon B. Easterbrook-Smith
3
and Geoffrey J. Howlett
1
1
Department of Biochemistry and Molecular Biology, The University of Melbourne, Victoria, Australia;
2
Department of Biological Sciences, University of Wollongong, NSW, Australia;
3
School of Molecular and Microbial Biosciences, University of Sydney, NSW, Australia
The effect of the extracellular chaperone, clusterin, on
amyloid fibril formation by lipid-free human apolipoprotein
C-II (apoC-II) was investigated. Sub-stoichiometric levels of
clusterin, derived from either plasma or semen, potently
inhibit amyloid formation by apoC-II. Inhibition is
dependent on apoC-II concentration, with more effective
inhibition by clusterin observed at lower concentrations of
apoC-II. The average sedimentation coefficient of apoC-II
fibrils formed from apoC-II (0.3 mgÆmL
)1
) is reduced by
coincubation with clusterin (10 lgÆmL
)1
). In contrast,
addition of clusterin (0.1 mgÆmL

)1
) to preformed apoC-II
amyloid fibrils (0.3 mgÆmL
)1
) does not affect the size
distribution after 2 days. This sedimentation velocity data
suggests that clusterin inhibits fibril growth but does not
promote fibril dissociation. Electron micrographs indicate
similar morphologies for amyloid fibrils formed in the
presence or absence of clusterin. The substoichiometric
nature of the inhibition suggests that clusterin interacts with
transient amyloid nuclei leading to dissociation of the
monomeric subunits. We propose a general role for clusterin
in suppressing the growth of extracellular amyloid.
Keywords: analytical ultracentrifugation; amyloid forma-
tion; nucleation; substoichiometric inhibition; protein
folding.
Human apoC-II (apoC-II) is mostly associated with plasma
very-low-density lipoproteins where it plays an important
physiological role as an activator of lipoprotein lipase [1,2].
When associated with polar lipids (e.g. phospholipids or
SDS) apoC-II adopts a primarily a helical conformation
[3–5]. However, under lipid-free conditions, apoC-II self-
associates in a time- and concentration-dependent manner
to form twisted ribbon-like fibrils with all the hallmarks of
amyloid [6]. These characteristics include the binding to
Congo Red with red-green birefringence under cross-
polarized light, binding to thioflavin T and increased
b structure [6]. The limited conformational stability of
apoC-II in the absence of lipid is typical of lipid-free

apolipoproteins and may underlie the general propensity of
apolipoproteins to form amyloid in vivo [7]. The ability of
apoC-II to form amyloid in vitro can be compared to in vivo
amyloid formation involving many of the exchangeable
apolipoproteins, including apoA-I [8,9], apoA-II [10,11],
apoA-IV [12], apoE [13], and apolipoprotein–like proteins,
a-synuclein [14] and serum amyloid A [15].
Amyloid formation by apoC-II provides a convenient
model to explore physiological parameters that modulate
amyloid formation in vivo. Previous studies suggest that the
chaperones Hsp27 and aB-crystallin suppress Ab amyloid
formation in vitro [16,17]. Our recent studies indicate that
a-crystallin inhibits amyloid formation at the nucleation
phase of amyloid formation, with no evidence of binding
to the apoC-II monomer or suppression of fibril elongation
[18]. In view of the extracellular location of amyloid
deposits, we investigated whether amyloid formation by
apoC-II is affected by the extracellular chaperone, clusterin
[19,20]. This protein is a disulfide-linked heterodimer, with
an average molecular mass of 75–80 kDa [19], and is
present in many biological tissues, with particularly high
concentrations present in plasma (35–105 lgÆmL
)1
)and
seminal fluid ( 400 lgÆmL
)1
) [21,22]. Clusterin is also
found in many amyloid-related lesions associated with
diseases including Alzheimer’s and atherosclerosis
[19,23,24]. Clusterin binds to the Alzheimer’s related

peptide Ab with nanomolar affinity, and inhibits its
aggregation [25]. Qualitative studies also point to an
inhibitory effect of clusterin on amyloid formation by Ab,
and a fragment of the prion protein [26,27]. In this
investigation, we explore the effect of clusterin on amyloid
formation by apoC-II and provide evidence that clusterin
acts at substoichiometric levels to inhibit amyloid nuclea-
tion and fibril extension.
MATERIALS AND METHODS
Materials
ApoC-II was expressed [28] and purified as described
previously [6,28]. ApoC-II was stored as a stock in 5
M
Correspondence to G. J. Howlett, Department of Biochemistry and
Molecular Biology, The University of Melbourne, Victoria 3010,
Australia.
Fax: + 61 39347 7730. Tel.: + 61 3 8344 7632,
E-mail:
Abbreviations: apo, apolipoprotein; GdnHCl, guanidine
hydrochloride; rmsd, root mean squared deviation.
Note: a website is available at
/>ghowlett_rp.htm.
(Received 13 December 2001, revised 19 April 2002,
accepted 25 April 2002)
Eur. J. Biochem. 269, 2789–2794 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02957.x
guanidine hydrochloride (GdnHCl) at a concentration of
40 mgÆmL
)1
. Clusterin was purified from human serum or
from human seminal fluid by immunoaffinity chromatog-

raphy as described previously [29]. Clusterin was stored at a
concentration of 1–1.4 mgÆmL
)1
in refolding buffer (10 m
M
sodium phosphate, 150 m
M
NaCl, 0.1% sodium azide,
pH 7.4) and kept at )20 °C until required. Experiments
were performed at 20 °C unless otherwise indicated.
Amyloid formation followed by thioflavin T reactivity
To follow time-dependent amyloid formation, apoC-II was
refolded by direct dilution into refolding buffer (0.3–
1mgÆmL
)1
) containing various concentrations of clusterin
or BSA. Control samples containing clusterin or BSA alone
were prepared in refolding buffer. GdnHCl was added to
provide the same concentration in all samples (160 m
M
). In
a microplate, aliquots containing 15 lg apoC-II, or 5 lg
protein for samples of BSA and clusterin alone, were added
to final solution volumes of 300 lL containing refolding
buffer and 5 l
M
thioflavin T. The fluorescence was
monitored using an f
max
fluorescence plate reader with a

444/485 nm excitation/emission filter set.
Analytical ultracentrifugation
Sample volumes of 300–400 lL were analyzed using the
XL-A analytical ultracentrifuge. Radial scans were taken in
continuous scanning mode and 0.002 cm radial increments.
For samples containing apoC-II aggregates, the sedimenta-
tion boundaries at different time points were analyzed to
obtain ls ) g*(s) sedimentation coefficient distributions. The
method is based on direct linear least-squares boundary
modeling by a superposition of sedimentation profiles of
ideal nondiffusing particles [30]. A regularization parameter
of p ¼ 0.95, and a buffer density of 1.01 gÆmL
)1
was used.
The nonsedimenting baselines at a rotor speed of 6000 r.p.m.
(2600 g), attributed to the presence of monomeric apoC-II,
was subtracted from the data by fitting a time-independent
absorbance background. A partial specific volume of
0.73 mLÆg
)1
was assumed based on the amino-acid compo-
sition of apoC-II. For samples of freshly prepared apoC-II
and for the slow moving boundary observed for incubated
apoC-II samples and mixtures of apoC-II and clusterin, data
were fitted to a model assuming a continuous size distribu-
tion [31]. For this analysis, the frictional ratio (f/f
0
)was
varied to give the lowest root mean squared deviation (rmsd)
for the sample containing freshly prepared apoC-II alone.

This best-fit value (f/f
0
¼ 1.75) was constrained for the
analyses of samples containing clusterin. A regularization
parameter of p ¼ 0.68 was used.
Electron microscopy
Solutions of apoC-II (0.3 mgÆmL
)1
) or clusterin
(0.1 mgÆmL
)1
) or mixtures containing both proteins, were
dilutedthreefoldinwaterandappliedtofreshlyglow-
discharged carbon-coated copper grids. After 1 min, excess
material was removed and the grids were washed twice with
20 lL water before negatively staining with 2% (w/v)
potassium phosphotungstate. The samples were imaged
using a JEOL 2000 transmission electron microscope
(Peabody, MA, USA) operating at 120 kV.
RESULTS
Sub-stoichiometric concentrations of clusterin inhibit
amyloid formation
Thioflavin T has low fluorescence in the presence of
monomeric apoC-II that increases proportionally to the
amount of apoC-II amyloid present [6,18]. This change in
fluorescence was used to monitor amyloid formation by
apoC-II in the presence of various concentrations of
clusterin (Fig. 1). For apoC-II alone, amyloid formation
occurs slowly over 9 days at room temperature. The
presence of increasing concentrations of serum clusterin

systematically reduces the accumulation of thioflavin T
reactive material, with near complete suppression of amy-
loid formation by 30 lgÆmL
)1
clusterin (Fig. 1A). Clusterin
alone (0.1 mgÆmL
)1
) shows no change in thioflavin T
reactivity over the same time course (data not shown).
Assuming a molecular mass of 8900 Da for apoC-II and
80 000 Da for clusterin, 0.3 mgÆmL
)1
apoC-II in the
presence of 30 lgÆmL
)1
clusterin represents a 90 : 1 molar
excess of apoC-II to clusterin. This stoichiometry of
inhibition is similar to the concentrations of a-crystallin
required to inhibit amyloid formation by apoC-II at a
Fig. 1. Time-dependent changes in amyloid formation by apoC-II
(0.3 mgÆmL
)1
). Amyloid formation was monitored using thioflavin T
fluorescence measurements for samples of apoC-II alone and in the
presence of serum clusterin (A) or seminal clusterin (B). ApoC-II alone
(d) and apoC-II in the presence of 1 lgÆmL
)1
(,), 10 lgÆmL
)1
(j),

30 lgÆmL
)1
(e), and 100 lgÆmL
)1
(m) clusterin.
2790 D. M. Hatters et al. (Eur. J. Biochem. 269) Ó FEBS 2002
concentration of 0.3 mgÆmL
)1
[18]. Similar levels of
inhibition were observed using seminal clusterin, with
0.1 mgÆmL
)1
providing complete inhibition of apoC-II
amyloid formation, equivalent to a 30 : 1 molar excess of
apoC-II to clusterin (Fig. 1B). The specificity of the
inhibition of amyloid formation by clusterin was assessed
in control experiments using BSA. The presence of BSA
(100 lgÆmL
)1
) had negligible effect on the time-dependent
formation of thioflavin T reactivity of 0.3 mgÆmL
)1
apoC-II
while BSA alone produced no changes in thioflavin T
reactivity over the same time (data not shown).
The substoichiometric concentrations of clusterin
required to suppress amyloid formation suggests that the
process is inhibited at the nucleation phase of fibril growth.
To investigate the kinetics in more detail, we performed
thioflavin T binding assays using a fixed molar ratio of

apoC-II to serum clusterin (90 : 1) but varied the total
protein concentration. Figure 2 shows the effects of varying
the concentration of apoC-II (0.3, 0.6 and 1.0 mgÆmL
)1
).
ApoC-II alone shows an increase in the rate of amyloid
formation as the apoC-II concentration is increased from
0.3 to 0.6 and 1 mgÆmL
)1
(Fig. 2). This accelerated rate of
amyloid formation is consistent with previous turbidity
studies [6]. Inhibition of apoC-II amyloid formation using a
fixed molar ratio of clusterin (1 : 90 ratio of clusterin:
apoC-II) is more complete at low apoC-II concentrations
(0.3 mgÆmL
)1
) compared to the inhibition observed at the
higher concentrations of apoC-II.
Amyloid fibril size is reduced by the presence of clusterin
The effect of clusterin on the aggregation-state of apoC-II
amyloid was investigated by sedimentation velocity analysis.
Sedimentation velocity data for apoC-II alone
(0.3 mgÆmL
)1
, incubated 9 days) reveals a fast-sedimenting
boundary comprising  65% of the absorbance (Fig. 3A).
This fast moving boundary is attributed to the presence of
high molecular mass amyloid fibrils [4,6]. Increasing the
rotor speed to 40 000 r.p.m. (116 200 g), revealed a slower
moving boundary. Analysis of this boundary yielded good

fits to a model describing a single sedimenting species with a
sedimentation coefficient of  1 S, and a molecular mass
10 000 Da. This is close to the expected mobility and
molecular mass of monomeric apoC-II [4,6]. The presence
of a distribution of large sedimenting species (centred at
 400 S) and monomeric apoC-II is consistent with a
bimodal population of high molecular mass amyloid and
monomers as previously observed [4,6,18].
The sedimentation velocity profile of 0.3 mgÆmL
)1
apoC-II incubated for 9 days in the presence of 10 lgÆmL
)1
serum clusterin is shown in Fig. 3B. The sedimentation data
shows a fast sedimenting boundary comprising  45% of
the absorbance. The absorbance contribution of clusterin is
negligible at these concentrations. At higher angular velo-
cities (40 000 r.p.m./116 200 g) the fast moving boundary
sedimented to the bottom of the cell revealing a slower
moving boundary indicative of predominately monomeric
apoC-II. It is noteworthy that the proportion of the fast
moving material relative to monomeric apoC-II is reduced
by the presence of clusterin (Fig. 3B compared to Fig. 3A),
consistent with the conclusion drawn from the data in
Figs 1 and 2 that clusterin inhibits apoC-II aggregation.
Fig. 2. Concentration dependence of amyloid formation by apoC-II in
the absence and presence of a fixed molar ratio of serum clusterin (90 : 1
molar ratio, apoC-II/clusterin). Data for apoC-II alone at 0.3 mgÆmL
)1
(d), 0.6 mgÆmL
)1

(r)and1.0mgÆmL
)1
(j). Data for apoC-II in the
presence of clusterin at apoC-II and clusterin concentrations of
0.3 mgÆmL
)1
and 0.03 mgÆmL
)1
(s), 0.6 mgÆmL
)1
and 0.06 mgÆmL
)1
(e)and1.0mgÆmL
)1
and 0.1 mgÆmL
)1
(h), respectively.
Fig. 3. Sedimentation velocity behaviour of apoC-II (0.3 mgÆmL
)1
)
after incubation for 9 days. (A) ApoC-II alone. (B) ApoC-II in the
presence of 10 lgÆmL
)1
serum clusterin. Radial scans are shown at
15 min intervals and a rotor speed of 6000 r.p.m. (2600 g)(thinblack
lines). Data were fitted to ls ) g*(s) analysis, with the fits shown as
thick grey lines. The optical density contribution due to clusterin is
negligible at these concentrations.
Ó FEBS 2002 Inhibition of apoC-II amyloidosis by clusterin (Eur. J. Biochem. 269) 2791
We fitted the data in Fig. 3A to a model describing

the sedimentation of a distribution of nondiffusing
particles [ls ) g*(s)] and obtained good fits (grey lines
in Fig. 3A) to a distribution of sedimentation coefficients
averaging 400 S (Fig. 4). ls ) g*(s) analysis of the data in
Fig. 3B produced good fits (Fig. 3B, grey lines) to a
sedimentation coefficient distribution averaging  180 S
(Fig. 4). This is significantly lower than the average for
apoC-II alone, suggesting that clusterin reduces the
overall size of the fibrils formed even though amyloid
formation remains highly cooperative.
While incubation of apoC-II in the presence of clusterin
reduces the size distribution of the amyloid fibrils (Fig. 4),
we wondered whether clusterin could alter the size of
preformed fibrils. Sedimentation velocity analysis was used
to compare the size distribution of apoC-II (0.3 mgÆmL
)1
)
incubated for 7 days at room temperature, followed by the
addition of seminal clusterin (0.1 mgÆmL
)1
) or an equivalent
volume of buffer and further incubation for 2 days.
ls ) g*(s) analysis of the sedimentation boundaries of
apoC-II alone produced a size distribution with a modal
sedimentation coefficient of 420 S, similar to that shown in
Fig. 4. Analysis of the incubated apoC-II sample containing
added clusterin produced a size distribution similar to
apoC-II alone, with a modal sedimentation coefficient of
450 S. Within the limits of experimental error, these results
indicate that clusterin does not affect the size of preformed

apoC-II amyloid fibrils.
Electron microscopy was used to characterize the mor-
phology of amyloid fibrils formed in the presence of
clusterin (Fig. 5). A sample of apoC-II (0.3 mgÆmL
)1
)
incubated in the presence of serum clusterin (0.1 mgÆmL
)1
)
for 10 days was analysed by negative staining with potas-
sium phosphotungstate (Fig. 5). The morphology of the
fibrils appeared indistinguishable to that of fibrils formed in
the absence of clusterin [6]. Distinctive features of clusterin
alone were not visible at the nominal magnification
(·40 000) used to visualize apoC-II amyloid. However,
single particles, attributed to clusterin, were observed at
higher magnifications (·100 000; data not shown). These
results suggest that despite the overall reduction in the mass
of amyloid formed, the fibril morphology was not altered by
the presence of clusterin.
Clusterin does not bind to apoC-II monomer
Clusterin has been reported to bind strongly to other
proteins, in particular, apoA-I and Ab [25,32]. Strong
binding could conceivably directly alter the kinetics of
amyloid formation. We used sedimentation velocity analysis
to monitor the state of association of freshly prepared
apoC-II and serum clusterin. Figure 6A shows the sedi-
mentation behaviour of serum clusterin alone at a rotor
speed of 40 000 r.p.m. 116 200 g. The scans (shown at
20 min intervals) reveal broad boundaries indicating a

heterogeneous population. Continuous sedimentation dis-
tribution analysis [31] resolved species ranging from 4 to
18 S, suggesting multimeric complexes of clusterin, consis-
tent with previous studies [19]. It must be emphasized,
however, that this analysis assumes noninteracting species
over the time period of the experiment, a condition that may
not strictly apply as previous studies using gel-filtration
chromatography indicate that oligomers of clusterin are in
rapid equilibrium [33]. The sedimentation of apoC-II alone
(freshly diluted from denaturant) produced scans that fitted
with random residuals to a single sharp peak (maximum
s ¼ 1 S), indicating the presence of close to 100% mono-
meric apoC-II (Fig. 6B). Sedimentation of the mixture of
apoC-II and clusterin produced boundaries that superim-
posed with a summation of the boundaries for the
sedimentation of apoC-II alone and clusterin alone
(Fig. 6C). This suggests that the sedimentation behaviour
of apoC-II and clusterin remain independent and that there
is negligible interaction between monomeric apoC-II and
clusterin.
Fig. 4. Sedimentation coefficient distributions for apoC-II
(0.3 mgÆmL
)1
) incubated in the presence and absence of serum clusterin
for 9 days. The ls ) g*(s) distributions correspond to the best fits of the
sedimentation velocity data presented in Fig. 3 [30]. ApoC-II alone
(solid line). ApoC-II and 10 lgÆmL
)1
serum clusterin (dashed line).
Fig. 5. Negatively stained transmission electron micrograph of apoC-II

fibrils (0.3 mgÆmL
)1
) formed in the presence of serum clusterin
(0.1 mgÆmL
)1
). Scale bar represents 200 nm.
2792 D. M. Hatters et al. (Eur. J. Biochem. 269) Ó FEBS 2002
DISCUSSION
Clusterin has a multitude of putative physiological functions
including controlling cell–cell and cell–substratum interac-
tions, regulating apoptosis and transporting lipids [20].
Recent evidence suggests that serum clusterin has a
chaperone activity similar to the small-heat-shock protein
family [19]. These studies show that clusterin inhibits the
aggregation of a variety of different proteins that are
partially denatured by reducing agents or heat-shock [19].
Suppression of aggregation of these denatured proteins
occurs at slightly substoichiometric molar ratios. Clusterin
has also been shown to inhibit the aggregation of amyloid-
ogenic peptides. A qualitative study, using transmission
electron microscopy, found that clusterin inhibited aggre-
gation of an amyloid forming peptide derived from the
prion protein at substoichiometric concentrations [26].
The aggregation of the Ab amyloid peptide after a fixed
time point with different concentrations of clusterin,
revealed that clusterin also inhibits Ab amyloid formation
[27].
Our results suggest that clusterin, purified from two
different tissue sources, suppresses amyloid formation at
substoichiometric concentrations (1 : 30–100, clusterin/

apoC-II). This behaviour is similar to our recent results
fortheeffectsofa-crystallin upon amyloid formation by
apoC-II [18]. Although the serum and seminal forms of
clusterin are encoded by the same structural gene, they are
known to differ in their glycosylation patterns [20]. Thus,
our finding that the serum and seminal forms of clusterin
exhibit similar chaperone activity suggests that the chaper-
one function of clusterin is not greatly affected by different
glycosylation patterns.
We propose that clusterin inhibits apoC-II amyloid
formation via a similar mechanism to a-crystallin [18]. This
mechanism postulates that clusterin interacts stoichiomet-
rically with amyloidogenic precursors (nuclei), leading to
dissociation of the nuclei back to monomer. At low apoC-II
concentrations clusterin is therefore capable of suppressing
amyloid formation substoichiometrically, because the con-
centration of amyloid nuclei is low. At higher apoC-II
concentrations, where self-association to form an oligomeric
nuclei is favored, clusterin is less effective in inhibiting
amyloid formation (Fig. 2). This mechanism would explain
the inhibition observed with other amyloid systems [26],
where nucleation is also rate-limiting [34].
The observation that clusterin reduces the sedimentation
coefficient distribution of apoC-II fibrils is significant. The
reduction in fibril size raises the possibility that clusterin also
inhibits fibril growth by interacting with the reactive ends of
fibrils. This action could either retard fibril extension or
cause dissociation of monomer subunits from the growing
fibril. Our sedimentation velocity data indicates that clus-
terin does not reduce the size of preformed fibrils. As nuclei

and fibril ends both recruit monomers, it seems likely that
their interfaces are structurally similar and consequently
they expose a similar binding site to clusterin. Because fibril
extension is much more rapid than nucleation rates, the
dominant mode of inhibition by clusterin may be on
nucleation, due to its rate limiting role in fibril growth. For
this reason, a bimodal population of monomers and large
polymers may persist, despite the retardation of fibril
growth.
Clusterin is localized to many amyloid deposits in vivo
and is expressed ubiquitously across many tissues and
species [20,23]. Clusterin is mostly located extracellularly
and is found localized to sites of injury (e.g. tissue damage
associated with ischemia, amyloid plaques, and cell necrosis
[20,23,35]). Such a biological distribution, in line with its
activity as a potent inhibitor of amyloid nucleation, suggests
that clusterin may play an active role in the regulation of
amyloid nucleation in both disease states and during normal
homeostasis.
Fig. 6. Sedimentation velocity analysis of 0.6 mgÆmL
)1
serum clusterin
(A), 0.3 mgÆmL
)1
freshly prepared apoC-II (B) and a mixture of apoC-II
and clusterin (C). Radial scans were acquired at 20-min intervals at a
rotor speed of 40 000 r.p.m. (116 200 g) [(A,B) solid lines; (C), points
(s)]. The solid lines in (C) represent the summation of the sedimen-
tation profiles of freshly prepared apoC-II and clusterin (A,B).
Ó FEBS 2002 Inhibition of apoC-II amyloidosis by clusterin (Eur. J. Biochem. 269) 2793

ACKNOWLEDGEMENTS
This work was supported by grants from the National Health and
Medical Research Council of Australia to G. J. H. and S. B. E., and
the Australian Research Council to S. B. E., and a Melbourne
Research Scholarship to D.M.H. We thank Lynne Lawrence at
C.S.I.R.O., Health Sciences and Nutrition, Parkvillle, Australia, for the
technical assistance with the electron microscopy.
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