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5.3 RESULTS
5.3.1 The effect of clusterin on amyloid formation measured by ThT assays
The effect of clusterin on proIAPP amyloid formation was investigated using ThT
assays. 250 µg/mL proIAPP was incubated in the absence and presence of 25, 50 and 100
µg/mL clusterin at 37 oC. These concentrations of clusterin are physiologically relevant as
clusterin is present in human serum at 35-105 µg/mL (Murphy et al., 1988). The amyloid
formation of proIAPP in the absence of clusterin showed a sigmoidal pattern as previously
described in Section 4.2.3. The presence of clusterin had no effect on the lag phase, which was
approximately 120 h, in the presence and absence of clusterin. In the absence of clusterin,
there was a rapid growth phase of approximately 50 h and by 170 h amyloid formation had
gone to completion. In the presence of clusterin, the growth phase was slowed in a dosedependent manner (Figure 5.2). In the presence of 25 µg/mL clusterin, amyloid formation had
not gone to completion by 340 h and the ThT fluorescence was approximately half that of
proIAPP in the absence of clusterin. Clusterin at higher concentrations could not prevent the
initiation of the growth phase but the growth phase appeared so slow as to have plateaued with
a low ThT fluorescence, indicating that very little amyloid had formed.
The inhibition of amyloid formation corresponded to molar ratios of 1 clusterin to 25100 proIAPP. These molar ratios are similar those seen over which clusterin inhibits amyloid
formation of apolipoprotein C II (1:90) (Hatters et al., 2002) and A42 (1:50) (Oda et al.,
1995). The molar ratio of clusterin to proIAPP also compares favourably with the minimal
molar ratios of clusterin to proteins, stressed by heat or reduction, that are required to almost
completely abolish their precipitation (Humphreys et al., 1999). For example the minimal
molar ratio of clusterin to glutathione-S-transferase required for clusterin to prevent the
reduction-induced precipitation of glutathione-S-transferase was approximately 1:3.2.
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Similarly the minimal molar ratios to inhibit the heat-induced precipitation for catalase, BSA
and -lactalbumin were 1:1.3, 1: 2.3 and 1:11 respectively (Humphreys et al., 1999).
ThT fluorescence (AU)
2500
2000
1500
1000
500
0
0
50
100
150 200
Time (h)
250
300
350
Figure 5.2 Clusterin inhibits proIAPP amyloid formation. 250 µg/mL proIAPP at 37 oC was
incubated in the absence of clusterin (red) and in the presence of 25 µg/mL (blue), 50 µg/mL (green)
and 100 µg/mL clusterin (black). Amyloid formation was measured using a ThT assays as described in
Section 2.2.16a.
The substoichiometric ratios over which clusterin suppresses proIAPP amyloid
formation suggest that clusterin is either interacting transiently with proIAPP or is interacting
with a small proportion of proIAPP species, such as the nuclei of amyloid formation. In this
context Hatters et al. have suggested that clusterin interacts with apolipoprotein C II nuclei
leading to the dissociation of the monomeric subunits (Hatters et al., 2002). It is not likely to
be the case for clusterin and proIAPP. If clusterin dissociated proIAPP nuclei, this would have
been as a longer lag phase as the time required for nucleation is prolonged. This was not
observed as the lag phase in the presence and absence of clusterin was the same, suggesting
that clusterin was unable to prevent the assembly of proIAPP monomers into a stable nucleus
(Figure 5.2). Clusterin slows the growth rate of amyloid formation in a dose-dependent
manner, suggesting that clusterin may inhibit elongation. Hatters et al. have suggested that
Chapter 5: The effects of clusterin on proIAPP amyloid formation and
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128
clusterin inhibits fibril growth because clusterin reduced the average sedimentation coefficient
of apolipoprotein C II fibrils (Hatters et al., 2002). Clusterin inhibits A amyloid formation
leading to the formation of soluble low molecular weight amyloidogenic oligomers that are
soluble and highly cytotoxic (Lambert et al., 1998). As low molecular weight oligomers do
not enhance ThT fluorescence (LeVine 1993), based on the ThT assay we cannot exclude the
possibility that the inhibition of proIAPP amyloid formation results in an increase in proIAPP
oligomers rather than fibrils.
5.3.2 Analysis of proIAPP and clusterin mixtures by transmission electron microscopy
TEM was used to characterise the morphology of amyloid fibrils formed in the
presence of clusterin. 500 µg/mL proIAPP in the presence of various concentrations of
clusterin up to 100 µg/mL was incubated for 10 days at 37 oC. At the end of 10 days, aliquots
were adsorbed to Formvar-coated copper grids, negatively stained with 1% phosphotungstic
acid and analysed by TEM. ProIAPP in the absence of clusterin showed typical fibril
morphology compared to that seen previously (Figure 5.3A and B, compare Figure 4.8). The
fibrils were approximately 10 nm wide and could be up to hundreds of nm long. The fibrils
also formed a net-like mesh (Figure 5.3A, E, G and H) as was previously described for
proIAPP (Figure 4.8) (Krampert et al., 2000). In the presence of 12.5 µg/mL clusterin,
proIAPP was fibrillar (Figure 5.3G) and looked very similar to the fibrils formed in the
absence of clusterin (Figure 5.3A). There were far fewer fibrils in the presence of 50 and 100
µg/mL clusterin, and very little evidence of amorphous aggregate (Figure 5.3E and F and
Figure 5.3C and D). This also indicated that the reduced ThT fluorescences of proIAPP seen
in the presence of clusterin were unlikely to be the result of the formation of amorphous
Chapter 5: The effects of clusterin on proIAPP amyloid formation and
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129
aggregate rather than fibrillar aggregate and were likely to be due to a suppression of amyloid
formation.
Small spherical structures were seen in proIAPP mixtures with 50 and 100 µg/mL
clusterin (Figure 5.3D and F, arrows). These spherical structures were seen in proIAPP
incubations in the absence of clusterin (Figure 4.10D) and may represent proIAPP oligomers.
Electron micrographs of clusterin alone also showed spherical structures of similar size
(Figure 5.18G), indicating these may be oligomeric clusterin but these were less numerous
than in the proIAPP and clusterin mixtures.
ProIAPP was also incubated with 100 µg/mL ovalbumin (Figure 5.3H) as a control for
a non-specific effect of a protein. ProIAPP has a molecular weight of 7.5 kDa and an
isoelectric point of 9.5. Clusterin is a glycoprotein with a molecular weight of around 75 kDa
and an isoelectric point of 4.5-5.2 (Murphy et al., 1988). At pH 7.4, an electrostatic interaction
may partially contribute to the suppression of amyloid formation therefore ovalbumin, as a
protein unrelated to amyloidosis, was chosen for its similar properties to clusterin: a relatively
large glycoprotein with a molecular weight of 45 kDa and an isoelectric point of 4.5-4.9.
Similar amounts of amyloid were formed in the presence of ovalbumin (Figure 5.3H)
compared to proIAPP alone (Figure 5.3A) indicating that the effect of clusterin on proIAPP
was unlikely to be non-specific.
1 µm
1 µm
Chapter 5: The effects of clusterin on proIAPP amyloid formation and
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(Figure on previous page)
130
Chapter 5: The effects of clusterin on proIAPP amyloid formation and
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131
Figure 5.3 Electron microscope images of proIAPP fibrils formed in the presence and absence of
clusterin. 500 µg/mL proIAPP was incubated for 10 days at 37 oC in PBSaz in the absence of clusterin
(A and B) and in the presence of 100 µg/mL (C and D), 50 µg/mL (E and F), and 12.5 µg/mL clusterin
(G). 500 µg/mL proIAPP was incubated with 100 µg/mL ovalbumin (H) as a non-specific protein.
Specimen were negatively stained with 1% (w/v) phosphotungstic acid and examined as described in
Section 2.2.19.
5.3.3 The binding of clusterin to freshly redissolved proIAPP measured by direct ELISA
The data presented above show that clusterin prolongs proIAPP in a soluble state,
preventing amyloid formation (Section 5.3.1 and 5.3.2). One way in which clusterin could
inhibit amyloid formation would be to interact with soluble amyloid-prone proIAPP species
and inhibit their conversion to amyloid. Therefore the binding of clusterin to freshly
redissolved proIAPP was measured to establish whether clusterin could bind to natively
unfolded proIAPP. The binding was measured by direct ELISA, in which one binding partner,
in this case proIAPP, was adsorbed to the ELISA plate and the other partner, in this case
clusterin was in solution.
The binding of clusterin to a variety of native and denatured proteins has been shown
to be enhanced at pH 5.5 compared to 7.4 (Hochgrebe et al., 2000). Clusterin was added at
various concentrations up to 100 µg/mL at pH 7.4 and 5.5. The unbound clusterin was then
washed away. Clusterin that was bound to the proIAPP was detected using a monoclonal
antibody against clusterin and a secondary antibody conjugated to horseradish peroxidase. The
amount of clusterin bound was proportional to the coloured product produced by horseradish
peroxidase and was quantified by its absorbance at 490 nm. Clusterin bound to proIAPP at
both pH 7.4 and 5.5 in a dose-dependent manner. The binding of clusterin to proIAPP did not
reflect non-specific binding as non-specifically bound clusterin was removed by washing with
0.1% (v/v) Triton X-100 in PBSaz prior to the detection of clusterin. Moreover, there was no
binding of clusterin to blocker only.
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The binding of clusterin to proIAPP was enhanced at pH 5.5 compared to 7.4, as has
been seen for other proteins. The increased binding of clusterin to proIAPP is likely to be due
to conformational changes in clusterin rather than proIAPP, which is adsorbed to the ELISA
tray. Clusterin is predominantly present in an oligomeric form at pH 7.4 but at pH 5.5 a greater
proportion of clusterin is present in a monomeric form (Hochgrebe et al., 2000). The increase
in monomer is associated with an increase in exposed hydrophobic surface (Hochgrebe et al.,
2000). This increase in exposed hydrophobic surface at pH 5.5 is likely to be linked to the
increase in activity as chaperones like clusterin mainly interact with their client proteins
through hydrophobic interactions.
0.45
Absorbance (492 nm)
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
20
40
60
80
100
120
Concentration of clusterin (µg/mL)
Figure 5.4 ELISA measurements of clusterin binding to proIAPP at pH 7.4 and 5.5. ProIAPP (100
µg/mL) was absorbed to an ELISA plate. The binding of clusterin in 1% BSA (w/v) in MES/PBSaz at
pH 7.4 (blue) or 5.5 (black) was measured as in Section 2.2.21. The data shown are means ± standard
deviations of triplicate measurements.
5.3.4 The binding of clusterin to freshly redissolved proIAPP by competition ELISA
ProIAPP is likely to undergo conformational changes prior to amyloid formation.
Soluble proIAPP is likely to comprise monomers and an increasing concentration of a
heterogenous assortment of oligomers, once going down the amyloid pathway. A time course
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133
experiment was therefore devised to measure the binding of clusterin to proIAPP at different
times along the amyloid formation pathway, as this would enable us to correlate the binding of
clusterin to proIAPP with the effect of clusterin on amyloid formation from it. A direct ELISA
protocol as described in Section 5.3.3, in which one binding partner, in this case proIAPP, is
adsorbed to the ELISA plate and the other binding partner in this case clusterin is in solution,
would not be suitable for a time course experiment. The assortment of proIAPP species are
likely to change with time of progression down the amyloidogenic pathway and these different
proIAPP species are unlikely to adsorb to an ELISA plate equally well. A change in the
amount of proIAPP adsorbed to the ELISA plate could be falsely interpreted as a change in the
extent of clusterin binding to proIAPP.
Detecting proIAPP bound to clusterin adsorbed to an ELISA tray would also pose
difficulties, as proIAPP-specific antibodies may not recognise different proIAPP species in the
amyloid formation pathway equally well. This has been shown in other amyloid systems: a
polyclonal antibody that has been raised against oligomeric A does not bind to monomeric
A or oligomers of A smaller than an octamer (Kayed et al., 2003). These changes in the
effectiveness of detecting bound proIAPP would also be falsely interpreted as a change in
clusterin binding to proIAPP. To avoid these possibilities, the binding of clusterin to proIAPP
was measured indirectly in a competition ELISA format.
In a competition ELISA, a protein (in this case clusterin) can either bind to a solution
phase ligand (in this case proIAPP) or solid phase ligand (adsorbed to the ELISA plate). The
binding of the protein (clusterin) to the solution phase ligand (proIAPP) is seen as a change in
the binding of clusterin to the solid phase ligand. The exact region(s) of the “chaperone active
site” of clusterin are not known, however ELISA studies suggest that the binding site for
native ligands is distinct from misfolded proteins (Lakins et al., 2002). Pre-incubation of
Chapter 5: The effects of clusterin on proIAPP amyloid formation and
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134
clusterin with native GST in solution inhibited the binding of clusterin to solid-phase GST in a
dose-dependent manner but did not inhibit the binding of clusterin to heat-stressed GST. In
addition, pre-incubation of clusterin with native A1-40 or heparin had no effect on the
binding of clusterin to lysozyme or insulin, stressed by reduction. As clusterin was considered
likely to bind to proIAPP at the same site as denatured proteins, denatured proteins were
chosen as competitors for clusterin binding to proIAPP.
Denatured lysozyme was initially chosen as the denatured target because it had been
previously characterised as a ligand for clusterin (Lakins et al., 2002) and its high isoelectric
point ~11 would reduce the likelihood of an electrostatic interaction with proIAPP, which has
a similarly high isoelectric point, ~9.5. Lysozyme was adsorbed to the plate and subsequently
denatured by reduction with 15 mM DTT. Clusterin was pre-incubated with freshly dissolved
proIAPP for 15 min prior to being applied to the denatured lysozyme in the ELISA plate and
incubated for a further 1 h. Clusterin was detected as previously described in Section 5.3.3.
^
1.4
rbance (492 nm)
1.2
#
1
*
*
0.8
0.6
^
#
^
Chapter 5: The effects of clusterin on proIAPP amyloid formation and
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135
Figure 5.5 Clusterin binding to denatured lysozyme in the presence of proIAPP. Lysozyme was
adsorbed to an ELISA plate and denatured by reduction with 15 mM DTT. Clusterin at 25 µg/mL (red
bars), 50 µg/mL (blue bars) or 100 µg/mL (green bars) was added to the wells in the presence of
proIAPP and clusterin in PBSaz, pH 7.4 that was bound to the denatured lysozyme was detected by
ELISA as described in Section 2.2.22. The data shown are means  standard deviations of triplicate
measurements. The significance of the amount of clusterin bound to denatured lysozyme in the absence
of proIAPP, compared to the amount bound in the presence of proIAPP was assessed using Student’s ttest (^ p < 0.05, * p < 0.005, # p < 0.0005).
In the absence of proIAPP, clusterin bound to denatured lysozyme in a dose-dependent
manner (Figure 5.5), consistent with previous findings (Lakins et al., 2002). At all fixed
concentrations of clusterin, proIAPP enhanced the binding of clusterin to denatured lysozyme
in a dose-dependent manner (Figure 5.5). At all concentrations of clusterin with 50, 100 and
200 µg/mL proIAPP, the amount of clusterin bound to denatured lysozyme was approximately
1.5-fold, 1.75-fold and 2.2-fold respectively compared to clusterin bound in the absence of
proIAPP (Figure 5.5). This indicated that the enhancement of clusterin binding to denatured
lysozyme was dependent on the concentration of proIAPP and independent of the
concentration of clusterin.
^
1
^
0.8
rbance (492 nm)
*
*
*
*
^
0.6
0.4
^
^
Chapter 5: The effects of clusterin on proIAPP amyloid formation and
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136
Figure 5.6 Clusterin binding to denatured lysozyme in 1% (w/v) BSA in the presence of
proIAPP. Lysozyme was adsorbed to an ELISA plate and denatured by reduction with 15 mM DTT.
Clusterin at 25 µg/mL (red bars), 50 µg/mL (blue bars) or 100 µg/mL (green bars) was added to the
wells in the presence of proIAPP and clusterin in 1% (w/v) BSA in PBSaz, pH 7.4 that was bound to
the denatured lysozyme was detected by ELISA as described in Section 2.2.22. The data shown are
means  standard deviations of triplicate measurements. The significance of the amount of clusterin
bound to denatured lysozyme in the absence of proIAPP, compared to the amount bound in the
presence of proIAPP was assessed using Student’s t-test (^ p < 0.05, * p < 0.005, # p < 0.0005).
One potentially artefactual reason for the proIAPP enhancement of clusterin binding to
denatured lysozyme may be that during the 15 min pre-incubation step, protein adsorbs nonspecifically to the tube walls. Therefore when clusterin is pre-incubated in the absence of
proIAPP more clusterin is adsorbed to tube walls and less is applied to the ELISA plate than
when clusterin and proIAPP are pre-incubated together. To exclude this possibility 1% (w/v)
BSA in PBSaz was used a diluent for clusterin and proIAPP mixtures. The enhancement of
clusterin binding to the denatured lysozyme in the presence of proIAPP was seen regardless of
the presence of 1% (w/v) BSA (Figure 5.6). This indicated that enhanced binding of clusterin
to denatured lysozyme in the presence of proIAPP was not an artefact of non-specific
adsorption losses of clusterin during the pre-incubation step.
5.3.5 The binding of clusterin to different denatured proteins in the presence of proIAPP
at pH 7.4
Another potentially artefactual reason for the enhanced binding of clusterin to
denatured lysozyme in the presence of proIAPP is that proIAPP could be binding to the
Chapter 5: The effects of clusterin on proIAPP amyloid formation and
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137
denatured lysozyme and making another surface for clusterin to attach to. Therefore, the
enhancement of clusterin binding to a denatured lysozyme in the presence of proIAPP was
investigated further to see if this effect was specific to lysozyme or to the mode of
denaturation. Lysozyme has a molecular weight of 14.3 kDa, a pI of around 11 and was
denatured in the ELISA experiments by reduction with 15 mM DTT. Denatured alcohol
dehydrogenase and insulin were chosen as solid-phase ligands for competition ELISA
experiments. Insulin has a molecular weight of 5.8 kDa, a pI of around 5.3 and was stressed by
reduction with 15 mM DTT. Alcohol dehydrogenase (ADH) has a molecular weight of 147
kDa, a pI of around 5.4-5.8 and was denatured by heating at 80 oC. Clusterin in 1% (w/v) BSA
was pre-incubated in the presence and absence of proIAPP at various concentrations and its
binding to denatured insulin or ADH monitored by competition ELISA as described above.
In the absence of proIAPP, clusterin bound to denatured insulin and ADH in a dosedependent manner (Figure 5.7) consistent with previous findings (Lakins et al., 2002).
ProIAPP enhanced the binding of clusterin to both denatured ADH and insulin at pH 7.4 in a
dose dependent manner. The binding of clusterin to denatured insulin was enhanced by
approximately 1.5-fold, 3-fold and 5-fold in the presence of 50 µg/mL, 100 µg/mL and 200
µg/mL respectively at all fixed concentrations of clusterin (Figure 5.7A). The binding of
clusterin to denatured ADH was enhanced by 2-fold, 3-fold and 4-fold in the presence of 50
µg/mL, 100 µg/mL and 200 µg/mL proIAPP respectively regardless of clusterin concentration
(Figure 5.7B). Thus, the observation of proIAPP-mediated enhancement of clusterin binding a
variety of denatured proteins that do not share similar size or charge properties or mode of
denaturation, suggests that proIAPP has a general effect on enhancing the binding of clusterin
to denatured proteins rather than proIAPP specifically binding to these different denatured
proteins and creating another surface for clusterin to attach to.
Chapter 5: The effects of clusterin on proIAPP amyloid formation and
cytotoxicity
A: Insulin, pH 7.4
B: ADH, pH 7.4
^
1.8
1.2
^
1.6
^ *
^
0.8
0.6
^
^ ^
^
0.4
Absorbance (492 nm)
1
Absorbance (492 nm)
138
1.4
1.2
1
^
0.8
0.6
0.4
0.2
0.2
0
0
50
100
200
Concentration of proIAPP (µg/mL)
0
0
50
100
200
Concentration of proIAPP (µg/mL)
Figure 5.7 Clusterin binding to denatured insulin and ADH in 1% (w/v) BSA in the presence of
proIAPP. Insulin (A) and ADH (B) were adsorbed to an ELISA plate and denatured by reduction with
15 mM DTT or heat respectively as described in Section 2.2.22 . Clusterin at 25 µg/mL (red bars), 50
µg/mL (blue bars) or 100 µg/mL (green bars) was added to the wells in the presence absence of various
concentrations of proIAPP in 1% BSA in PBSaz, pH 7.4. Clusterin that was bound to the denatured
protein was detected by ELISA as described in Section 2.2.22. The data shown are means  standard
deviations of triplicate measurements. The significance of the amount of clusterin bound to denatured
protein target in the presence of proIAPP, compared amount bound in the absence of proIAPP was
assessed using Student’s t-test (^ p < 0.05, * p < 0.005, # p < 0.0005).
One explanation for the enhanced binding of clusterin to denatured proteins in the
presence of proIAPP could be that clusterin binds to both the denatured protein and proIAPP.
If clusterin binds simultaneously to the denatured protein and proIAPP, the effect of proIAPP
may be to cross-link clusterin bound to the denatured protein (Figure 5.8B). Arguing against
this proposition is the fact that the enhancement of clusterin binding to a denatured protein is
not dependent on the clusterin:proIAPP ratio as would be expected for a clusterin-proIAPP
complex. 25, 50 or 100 µg/mL clusterin and 200 µg/mL proIAPP corresponds to molar
clusterin:proIAPP ratios of 1:80, 1:40 and 1:20 yet this concentration of proIAPP enhances
clusterin binding to denatured lysozyme by approximately 2.2-fold regardless of clusterin
concentration (Section 5.3.4). If clusterin binds proIAPP and denatured protein, an
enhancement due to cross-linking would be more consistent with the enhancement being
Chapter 5: The effects of clusterin on proIAPP amyloid formation and
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139
dependent on the clusterin: proIAPP ratio rather than dependent on the proIAPP concentration
and independent of the clusterin concentration.
A
B
Figure 5.8 Diagram of how proIAPP may cross-link clusterin bound to denatured protein. Panel
A shows clusterin (blue circles) in the absence of proIAPP bound to the adsorbed denatured protein
(red lines) and the clusterin detected by the clusterin monoclonal antibodies ( ). Panel B shows that
proIAPP (yellow crosses) may cross-link clusterin bound to the denatured lysozyme.
Another possibility is that the binding of proIAPP to clusterin results in the generation
of an altered form of clusterin with an enhanced ability to bind to the denatured proteins
(Figure 5.9). There is a precedent for this proposal: the activity of clusterin is pH-dependent,
with stronger binding to client proteins at pH 5.5 compared to 7.4 (Hochgrebe et al., 2000).
This increase in activity is associated with a shift from a predominantly oligomeric clusterin to
a more monomeric form and an increase in exposed hydrophobic surface. The increase in
exposed hydrophobic surface with decrease in pH is likely to be linked to the increase in
activity as chaperones mainly interact with misfolded substrates through hydrophobic
interactions. ProIAPP may induce a similar increase in clusterin activity by increasing the
proportion of monomeric clusterin as occurs with a decrease in pH. Freshly dissolved proIAPP
is a natively unfolded protein (Section 4.2.1) and may binding in the putative “native ligand
site” of clusterin, suggested by Lakins et al., forming a complex with an enhanced activity and
which is still able to bind to denatured ligands through its putative “denatured ligand site”
(Figure 5.9A) (Lakins et al., 2002). However the proIAPP enhancement of clusterin binding to
Chapter 5: The effects of clusterin on proIAPP amyloid formation and
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140
denatured proteins is not dependent on the clusterin:proIAPP ratio, with which such a
mechanism would be expected to be dependent. Alternatively proIAPP may interact weakly or
transiently with clusterin, which may disrupt oligomeric clusterin and shift it to a more
monomeric form (Figure 5.9B). Freshly redissolved proIAPP also has characteristics of an
intrinsic pre-molten globule (Section 4.2.1) meaning that it contains some residual secondary
structure but less than a molten globule and is also able to weakly bind bis-ANS, indicating
that it has some exposed hydrophobic patches (Section 4.2.9). ProIAPP may transiently bind
to clusterin through these exposed hydrophobic patches but not exhibit sufficient exposed
hydrophobic surface to form stable complexes. Such a mechanism may not be dependent on
the clusterin:proIAPP ratio and could be dependent on the proIAPP concentration.
A
B
Figure 5.9 Diagram of possible ways in which proIAPP induces clusterin to shift from an
oligomeric state to monomeric state. Panel A shows a proIAPP (yellow crosses) disrupts oligomeric
clusterin (blue circles) by forming a complex that is able to bind to denatured proteins (red lines). Panel
B shows proIAPP interacts transiently with clusterin, disrupting oligomeric clusterin and increasing the
proportion of clusterin in a monomeric state. Clusterin monoclonal antibodies are represented by ( ).
5.3.6 The binding of clusterin to different denatured proteins in the presence of proIAPP
at pH 5.5
Chapter 5: The effects of clusterin on proIAPP amyloid formation and
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If the effect of proIAPP is to make clusterin more active through a similar mechanism
to that arising from a decrease in pH, then this enhancement should be reduced at pH 5.5
compared to pH 7.4 because clusterin is more active at this pH. Therefore to investigate this
possibility the binding of clusterin to denatured target proteins was tested at pH 5.5 in the
presence of various concentrations of proIAPP.
In the absence of proIAPP, clusterin at pH 5.5 bound to denatured lysozyme, insulin
and ADH in a concentration-dependent manner (Figure 5.10). The amount of clusterin bound
to all the denatured proteins in the absence of proIAPP was increased at pH 5.5 compared to
pH 7.4 (compare Figure 5.10A, 5.10B and 5.10C with Figure 5.6, 5.7A and 5.7B). At pH 5.5,
there was no enhancement of clusterin binding to the denatured ADH or insulin in the
presence of proIAPP, at any of the proIAPP concentrations tested (Figure 5.10).
Rather, in the presence of proIAPP at pH 5.5, clusterin binding to denatured lysozyme,
insulin and ADH exhibited a complicated pattern but the same trend was seen for all the
proteins (Figure 5.10). The amount of clusterin bound to denatured insulin and ADH was
reduced in the presence of 50 µg/mL proIAPP compared to clusterin in the absence of
proIAPP. At 100 µg/mL and 200 µg/mL proIAPP, more clusterin was bound to the denatured
insulin and ADH than in the presence of 50 µg/mL proIAPP but there was still less bound than
in the absence of proIAPP. There was less of an effect for proIAPP and clusterin binding to
denatured lysozyme at pH 5.5. However at 25 and 50 µg/mL clusterin, the presence of 50
µg/mL or 100 µg/mL proIAPP reduced the amount of clusterin bound to denatured lysozyme
as seen for denatured insulin and ADH. More clusterin bound to denatured lysozyme for 25
µg/mL and 50 µg/mL clusterin in the presence of 200 µg/mL proIAPP than at 50 µg/mL or
100 µg/mL proIAPP, as seen for denatured insulin or ADH at pH 5.5. This is different to the
progressive decrease in clusterin being bound to the denatured proteins with increased
Chapter 5: The effects of clusterin on proIAPP amyloid formation and
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142
proIAPP concentration that would be expected if the only effect is that more clusterin is bound
to proIAPP.
The results at pH 5.5 in the presence of proIAPP are likely to be the net result of
proIAPP-mediated enhancement and competition between proIAPP and the denatured protein
target for clusterin binding. At pH 5.5 clusterin is more active than at pH 7.4, therefore it
would require a higher concentration of proIAPP to increase the activity of clusterin. At pH
5.5 the binding of clusterin to proIAPP is increased so there is likely to be less clusterin
available for binding to the denatured proteins. These two competing effects are likely to
produce the reduction in clusterin bound to denatured proteins in the presence of 50 µg/mL
proIAPP compared to the absence of proIAPP and the dose-dependent increase in the amount
of clusterin bound to the denatured proteins in the presence of at 100 µg/mL and 200 µg/mL
proIAPP compared to 50 µg/mL proIAPP.
A: Lysozyme, pH 5.5
B: Insulin, pH 5.5
1.4
^
^
^
^
2
nm)
nm)
1.2
1
2.5
^
Chapter 5: The effects of clusterin on proIAPP amyloid formation and
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143
Figure 5.10 Clusterin binding to denatured proteins in 1% (w/v) BSA in MES/PBSaz, pH 5.5 in
the presence and absence of proIAPP. Lysozyme, (A), insulin (B and ADH (C were adsorbed to
ELISA plates. Lysozyme and insulin were denatured by reduction using DTT and ADH was denatured
with heat as described in Section 2.2.22. Clusterin at 25 µg/mL (red bars), 50 µg/mL (blue bars) or
100 µg/mL (green bars) was added to the wells in the presence or absence of proIAPP at MES/PBSaz,
pH pH 5.5 and clusterin that was bound to the denatured target proteins was detected by ELISA as
described in Section 2.2.22. The data shown are means  standard deviations of triplicate
measurements. The significance of the amount of clusterin bound to denatured protein in the absence
of proIAPP, compared to the amount bound in the presence of proIAPP was assessed using Student’s ttest (^ p < 0.05, * p < 0.005, # p < 0.0005).
5.3.7 Time course of binding of clusterin to denatured lysozyme in the presence of
proIAPP
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Having characterised the competition ELISA for measuring the interaction between
clusterin and freshly dissolved proIAPP, this assay was used to measure the interaction
between clusterin and proIAPP as proIAPP progresses down the amyloid forming pathway.
ProIAPP was freshly redissolved to 500 µg/mL and then incubated at 37 oC. Aliquots of
proIAPP were removed from this incubating solution at 0, 22, 44 and 72 h. BCA assays
showed that the proIAPP concentration remained the same over 72 h, after which proIAPP
precipitated. Clusterin was pre-incubated with proIAPP for 15 min prior to being applied to
the denatured lysozyme in the ELISA plate and incubated for a further 1 h. Clusterin bound to
the denatured lysozyme was detected as previously described in Section 5.3.4.
As previously discussed, the enhancement of clusterin binding to denatured lysozyme
by freshly redissolved proIAPP is dependent on the concentration of proIAPP (Section 5.3.4).
This enhancement was also found to be dependent on the progression of proIAPP along the
amyloid-forming pathway prior to being mixed with clusterin. This enhancement was greatest
with freshly dissolved proIAPP and decreased with time down the amyloidogenic pathway.
Regardless of clusterin concentration, freshly redissolved 200 µg/mL proIAPP enhanced
clusterin binding to denatured lysozyme by approximately 2.2-fold (Figure 5.5 and Figure
5.11A, 0 h). After proIAPP had been incubated for 22 h, 200 µg/mL proIAPP only enhanced
clusterin binding to denatured lysozyme by approximately 1.8-fold (Figure 5.11A, 22 h). After
proIAPP had been incubated for 44 or 72 h, there was no difference in the amount of clusterin
bound to denatured lysozyme in the presence 200 µg/mL proIAPP or in the absence of
proIAPP (Figure 5.11A, 44 and 72 h). The same incubation time-dependent decrease in
proIAPP enhanced clusterin binding to denatured lysozyme was seen at 50 and 100 µg/mL
proIAPP (Figure 5.11B and C). In the presence of 50 µg/mL and 100 µg/mL proIAPP,
clusterin binding to denatured lysozyme was enhanced by freshly redissolved proIAPP by 1.5-
Chapter 5: The effects of clusterin on proIAPP amyloid formation and
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145
fold and 1.8-fold respectively. After proIAPP had been incubated for 44 or 72 h, there was no
longer an enhancement of clusterin binding to denatured lysozyme (Figure 5.11B and C). The
interaction between soluble proIAPP and clusterin was not measured after 72 h incubation as
there was no longer sufficient proIAPP remaining in solution. The fact that there was no
change in the amount of clusterin bound to the denatured lysozyme in the presence or absence
of proIAPP at 44 or 72 h can be interpreted either as the enhancement of clusterin activity by
the aged proIAPP species is equal to the amount of clusterin bound to proIAPP or that
proIAPP species present at 44 and 72 h do not interact with clusterin. One of the hypotheses
proposed for why amyloid formation occurs even though chaperones may be present, is that
the partly folded intermediate that is sufficiently structured and does not display significant
exposed hydrophobic surface for it to be detected by the chaperone machinery (Section 1.11).
300
^* ^
250
^ * ^
200
B: 100 µg/mL proIAPP
to denatured lysozyme
to denatured lysozyme
A: 200 µg/mL proIAPP
300
250
^ # #
200
*
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146
Figure 5.11 The effect of proIAPP on the binding of clusterin to denatured lysozyme as proIAPP
proceeds down the amyloid forming pathway. 500 µg/mL proIAPP was incubated at 37 oC for 72 h.
At 0, 22, 44 and 72 h the soluble proIAPP concentration was measured by BCA assay (Section 2.2.15).
Soluble proIAPP at concentrations of 200 µg/mL (A), 100 µg/mL (B) or 50 µg/mL (C) was mixed with
25 µg/mL (red bars), 50 µg/mL (blue bars) or 100 µg/mL (green bars) clusterin in PBSaz, pH 7.4 and
pre-incubated for 15 min, after which the clusterin and proIAPP mixtures were applied to denatured
lysozyme adsorbed to the ELISA plate. Clusterin in the absence of proIAPP was considered 100%
binding of clusterin to the denatured lysozyme. Clusterin bound to denatured lysozyme was detected as
described in Section 2.2.22. The data shown are means  standard deviations of triplicate
measurements. The significance of the amount of clusterin bound to denatured protein in the absence
of proIAPP, compared to the amount bound in the presence of proIAPP was assessed using Student’s ttest (^ p < 0.05, * p < 0.005, # p < 0.0005).