Amyloid formation followed by thioflavin T reactivity To followtime-dependent amyloid formation, apoC-II was refolded by direct dilution into refolding buffer 0.3– 1 mgÆmL1 containing va
Trang 1Suppression of apolipoprotein C-II amyloid formation
by the extracellular chaperone, clusterin
Danny M Hatters1, Mark R Wilson2, Simon B Easterbrook-Smith3and Geoffrey J Howlett1
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; nucleaforma-tion; substoichiometric inhibiforma-tion; 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 viewof 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
M A T E R I A L S A N D M E T H O D S
Materials ApoC-II was expressed [28] and purified as described previously [6,28] ApoC-II was stored as a stock in 5
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: ghowlett@unimelb.edu.au
Abbreviations: apo, apolipoprotein; GdnHCl, guanidine
hydrochloride; rmsd, root mean squared deviation.
Note: a website is available at
http://www.biochemistry.unimelb.edu.au/bch/research/
ghowlett_rp.htm.
(Received 13 December 2001, revised 19 April 2002,
accepted 25 April 2002)
Trang 2guanidine 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)1in refolding buffer (10 mM
sodium phosphate, 150 mM NaCl, 0.1% sodium azide,
pH 7.4) and kept at )20 C until required Experiments
were performed at 20C unless otherwise indicated
Amyloid formation followed by thioflavin T reactivity
To followtime-dependent amyloid formation, apoC-II was
refolded by direct dilution into refolding buffer (0.3–
1 mgÆmL)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 mM) 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 lM thioflavin T The fluorescence was
monitored using an fmaxfluorescence 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)1was 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)1was assumed based on the amino-acid
compo-sition of apoC-II For samples of freshly prepared apoC-II
and for the slowmoving 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 fricdistribu-tional ratio (f/f0) w as
varied to give the lowest root mean squared deviation (rmsd)
for the sample containing freshly prepared apoC-II alone
This best-fit value (f/f0 ¼ 1.75) was constrained for the
analyses of samples containing clusterin A regularization
parameter of p¼ 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
diluted threefold in w ater and applied to freshly glow
-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
R E S U L T S
Sub-stoichiometric concentrations of clusterin inhibit amyloid formation
Thioflavin T has lowfluorescence 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)1clusterin (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)1clusterin 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.
Trang 3concentration 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)1apoC-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 lowapoC-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) and 1.0 mgÆmL)1(j) Data for apoC-II in the
presence of clusterin at apoC-II and clusterin concentrations of
0.3 mgÆmL)1and 0.03 mgÆmL)1(s), 0.6 mgÆmL)1and 0.06 mgÆmL)1
(e) and 1.0 mgÆmL)1and 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)1serum clusterin Radial scans are shown at
15 min intervals and a rotor speed of 6000 r.p.m (2600 g) (thin black 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.
Trang 4We 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
s¼ 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)1serum 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.
Trang 5D I S C U S S I O N
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 showthat 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 for the effects of a-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 lowapoC-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)1serum clusterin
(A), 0.3 mgÆmL)1freshly 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).
Trang 6A C K N O W L E D G E M E N T S
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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