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Tài liệu Báo cáo khoa học: Affinity and kinetics of proprotein convertase subtilisin ⁄ kexin type 9 binding to low-density lipoprotein receptors on HepG2 cells docx

Affinity and kinetics of proprotein convertase
subtilisin

kexin type 9 binding to low-density lipoprotein
receptors on HepG2 cells
Seyed A. Mousavi
1
, Knut E. Berge
1
, Trond Berg
2
and Trond P. Leren
1
1 Unit for Cardiac and Cardiovascular Genetics, Department of Medical Genetics, Oslo University Hospital Rikshospitalet, Norway
2 Department of Molecular Biosciences, University of Oslo, Norway
Keywords
association; dissociation; dissociation
constants; low-density lipoprotein receptor;
proprotein convertase subtilisin ⁄ kexin 9
Correspondence
T. P. Leren, Unit for Cardiac and

Cardiovascular Genetics, Department of
Medical Genetics, Oslo University Hospital
Rikshospitalet, P.O. Box 4950 Nydalen,
NO-0424 Oslo, Norway
Fax: +47 23075561
Tel: +47 23075552
E-mail: trond.leren@rikshospitalet.no
(Received 29 March 2011, revised 7 June
2011, accepted 16 June 2011)
doi:10.1111/j.1742-4658.2011.08219.x
Proprotein convertase subtilisin ⁄ kexin type 9 (PCSK9) is a secreted protein
that regulates the number of cell surface low-density lipoprotein receptors
(LDLRs) and the levels of low-density lipoprotein cholesterol in plasma.
Intact cells have not previously been used to determine the characteristics
of binding of PCSK9 to LDLR. Using PCSK9 iodinated by the tyramine
cellobiose (TC) method ([
125
I]TC-PCSK9), we measured the affinity and
kinetics of binding of PCSK9 to LDLR on HepG2 cells at 4 °C. The extent
of [
125
I]TC-PCSK9 binding increased as cell surface LDLR density
increased. Unlabeled wild-type and two gain-of-function mutants of
PCSK9 reduced binding of [
125
I]TC-PCSK9. The Scatchard plot of the
binding-inhibition curve was curvilinear, indicative of high-affinity and
low-affinity sites for PCSK9 binding on HepG2 cells. Nonlinear regression
analysis of the binding data also indicated that a two-site model better fit-
ted the data. The time course of [
125
I]TC-PCSK9 binding showed two
phases in the association kinetics. Dissociation of [
125
I]TC-PCSK9 also
occurred in two phases. Unlabeled PCSK9 accelerated the dissociation of
[
125
I]TC-PCSK9. At low pH, only one phase of dissociation was apparent.
Furthermore, the dissociation of [


125
I]TC-PCSK9 under pre-equilibrium
conditions was faster than under equilibrium conditions. Overall, the data
suggest that PCSK9 binding to cell surface LDLR cannot be described by
a simple bimolecular reaction. Possible interpretations that can account for
these observations are discussed.
Introduction
Proprotein convertase subtilisin ⁄ kexin type 9 (PCSK9)
is a protein secreted by the liver that was recognized as
an important regulator of cholesterol homeostasis
through its link to autosomal dominant hypercholester-
olemia [1–3]. Central to its role as a cholesterol-regula-
tory protein is the ability of PCSK9 to downregulate the
low-density lipoprotein (LDL) receptor (LDLR) [4–6].
Hepatic LDLR seems to be particularly susceptible to
this effect of PCSK9. PCSK9-mediated downregulation
of hepatic LDLR inhibits LDL uptake from plasma,
thus increasing the concentrations of LDL cholesterol
in plasma [4,7]. Besides the effect on hepatic LDLR
levels, endocytosis of PCSK9 by the liver is also
responsible for clearance of PCSK9 from the circulation
[7].
The importance of PCSK9 in maintaining choles-
terol homeostasis is clinically evident, in that PCSK9
gain-of-function mutations are associated with elevated
Abbreviations
ECD, extracellular domain; EGF-A, epidermal growth factor-like repeat A; LDL, low-density lipoprotein; LDLR, low-density lipoprotein
receptor; LPDS, lipoprotein-depleted serum; PCSK9, proprotein convertase subtilisin ⁄ kexin type 9; PCSK9-WT, wild-type proprotein
convertase subtilisin ⁄ kexin type 9; TC, tyramine cellobiose.
2938 FEBS Journal 278 (2011) 2938–2950 ª 2011 The Authors Journal compilation ª 2011 FEBS
levels of plasma LDL cholesterol, whereas loss-of-func-
tion mutations are associated with low levels of plasma
LDL cholesterol [1,8–12]. Further data supporting the
importance of PCSK9 in cholesterol homeostasis come
from studies in mice, demonstrating that overexpres-
sion of PCSK9 in the liver results in increased plasma
LDL cholesterol levels, whereas knockout of the
PCSK9 gene decreases plasma levels of LDL choles-
terol [4–6,13] (for a recent review see [14]).
PCSK9 exerts its action by interacting with a bind-
ing site within the epidermal growth factor-like
repeat A (EGF-A) of LDLR [15]. The EGF-A-binding
site of PCSK9 has been shown to be composed of resi-
dues within the catalytic domain [16,17]. Recent data
also indicate the potential involvement of other sites in
PCSK9 in LDLR binding [18].
Many of the data on parameters that describe
PCSK9 binding to LDLR have been obtained from
Biacore surface plasmon reasonance studies, using a
purified extracellular domain (ECD) of the LDLR.
However, although sensitive and powerful, this
method may not completely reflect the situation of
membrane-embedded, full-length LDLR in intact cells.
In this report, we present studies on the interaction of
PCSK9 with LDLR on HepG2 cells, a cell line that is
widely used to study PCSK9-mediated degradation of
LDLR.
Results
Specificity of PCSK9 iodinated by the tyramine
cellobiose (TC) method ([
125
I]TC-PCSK9) binding
to HepG2 cells
Incubation of cells in lipoprotein-free medium increa-
sed the level of LDLR expressed on the cell surface
 1.8-fold (1.82 ± 0.10, n = 3) as compared with cells
that had been grown for 48 h in complete growth med-
ium (Fig. 1A). Under both conditions, the amount of
[
125
I]TC-PCSK9 specifically bound was linearly related
to cell density. Incubation of cells in lipoprotein-free
medium was also associated with a  1.6-fold increase
(1.64 ± 0.16, n = 3) in the binding of [
125
I]TC-PCSK9
(Fig. 1B). The binding of [
125
I]TC-PCSK9-D374Y is
presented for comparison. Approximately five times
less [
125
I]TC-PCSK9-D374Y than [
125
I]TC-PCSK9 was
needed to achieve equivalent binding (1.7 ± 0.07,
n = 3) (Fig. 1C), which is consistent with the higher
affinity of PCSK9-D374Y for LDLR at neutral pH
(see below). The extent of binding to blank wells was
similar for both ligands, and the radioactivity asso-
ciated with cells was at least 10 times of the counts
associated with blank wells.
Fig. 1. Cell density dependence of the binding of [
125
I]TC-PCSK9
and [
125
I]TC-PCSK9-D374Y to HepG2 cells. Varying numbers of
HepG2 cells were grown in complete growth medium (lower
curves) or LPDS-containing Opti-MEM (upper curves), as described
in Experimental procedures. The relative amount of cell-surface
immunoreactive LDLR (A), as measured by the amount of specific
binding of
125
I-labeled anti-(rabbit IgG) to cells, and the specific
binding of [
125
I]TC-PCSK9 (5 lgÆmL
)1
,70nM) (B) and [
125
I]TC-
PCSK9-D374Y (1 lgÆmL
)1
,14nM) (C) to cells were determined as
described in Experimental procedures. Mean cell numbers in wells
seeded with the highest cell density were 7.55 · 10
5
and 7.5 · 10
5
for cells grown in complete growth medium and LPDS-containing
Opti-MEM, respectively. The numbers of cells in wells with lower
cell numbers could not be reliably determined. The results are
means ± standard deviations of triplicate determinations from a sin-
gle experiment. Similar results were obtained in several indepen-
dent single-point binding experiments at high cell density, each
performed in duplicate. The values given in the text are the
means ± standard deviations of three independent experiments
performed at high cell density, including the results obtained with
the highest cell density in this experiment.
S. A. Mousavi et al. Characterization of the binding of PCSK9 to intact cells
FEBS Journal 278 (2011) 2938–2950 ª 2011 The Authors Journal compilation ª 2011 FEBS 2939
As the gain-of-function D374Y mutation is localized
in the LDLR-binding region of PCSK9, the enhanced
binding of [
125
I]TC-PCSK9-D374Y to HepG2 cells can
be attributed entirely to its higher affinity for LDLR.
We therefore conclude that LDLR is the main receptor
responsible for binding of [
125
I]TC-PCSK9 and
[
125
I]TC-PCSK9-D374Y to HepG2 cells.
To further demonstrate the specificity of [
125
I]TC-
PCSK9 binding, we incubated the cells for 30 min at
4 °C in the presence of different unlabeled ligands
prior to incubating the cells with [
125
I]TC-PCSK9. The
inclusion of a 200-fold excess of unlabeled PCSK9-
D374Y reduced binding by 82–90% (depending on the
batch used). Unlabeled wild-type PCSK9 and PCSK9-
S127R were also able to reduce the binding of
[
125
I]TC-PCSK9 (see below). TLDLR possesses distinct
binding domains for apoB-100 (the main apolipopro-
tein in LDL) and PCSK9. In agreement with previous
studies showing that LDL can inhibit the uptake
of PCSK9 in cells [19,20], binding of [
125
I]TC-PCSK9
was inhibited by > 60% when unlabeled LDL
(1 mgÆmL
)1
) was included in the incubation medium
(data not shown), presumably because of steric block-
ing of the adjacent EGF-A domain. Incubation with
formaldehyde-treated BSA at a concentration sufficient
to saturate scavenger receptors (1 mgÆmL
)1
) [21] had
little effect on specific [
125
I]TC-PCSK9 binding to
HepG2 cells (data not shown), further supporting the
specificity of the binding.
Estimation of binding affinity of wild-type and
two mutant variants of PCSK9
In preliminary saturation experiments, we found that it
was very difficult to achieve complete saturation curves
for [
125
I]TC-PCSK9 binding to HepG2 cells. More-
over, large amounts of unlabeled PCSK9 (wild type
and D374Y) were required to determine nonspecific
binding. These technical limitations precluded determi-
nation of equilibrium dissociation constants (K
d
) for
[
125
I]TC-PCSK9 and [
125
I]TC-PCSK9-D374Y.
In order to estimate the binding affinities of PCSK9
and PCSK9-D374Y for HepG2 cell LDLR, we incu-
bated the cells with a fixed concentration of [
125
I]
TC-labeled ligand in the presence of increasing concen-
trations of the unlabeled counterpart (Fig. 2B,C). The
abilities of unlabeled PCSK9-D374Y and PCSK9-
S127R to reduce [
125
I]TC-PCSK9 binding were also
compared with that of unlabeled wild-type PCSK9
(Fig. 2A). The binding data were analyzed either by
nonlinear regression analysis or by the method of Scat-
chard. Nonlinear regression analysis of data from indi-
vidual binding curves indicated that the data were
described best by a two-binding site model. The IC
50
(the concentration of the competing ligand that inhib-
its 50% of the specific binding of [
125
I]TC-PCSK9) val-
ues for the higher-affinity and lower-affinity sites are
shown in Table 1, where it can be seen that binding is
inhibited most effectively by unlabeled PCSK9-D374Y.
Unlabeled PCSK9-D374Y was also able to reduce the
binding of its labeled counterpart ([
125
I]TC-PCSK9-
D374Y) to HepG2 cells in a concentration-dependent
manner (Fig. 2C; Table 1). The relative proportions of
the two binding sites were roughly equal.
Analysis of the same binding data by the Scatchard
method yielded concave upward curves (Fig. 2B,C,
insets), suggesting the presence of two binding sites ⁄
states. The K
d
values obtained for the higher-affinity
and lower-affinity binding sites are listed in Table 1.
The high-affinity and the low-affinity sites accounted
for 25% and 75% of the total binding, respectively.
These estimates of the relative proportions of sites are
different from those estimated by nonlinear regression
analysis. However, it has been well established that the
Scatchard method is sensitive to slight experimental
errors, making accurate estimates of the number of
binding sites from Scatchard plots difficult [22,23].
The higher-affinity and lower-affinity sites for
PCSK9 binding on HepG2 cells may be indicative of
either the presence of two subpopulations of LDLR
with different affinities for PCSK9, or negative cooper-
ativity among interacting LDLRs, although other
explanations are also possible (see below). The Hill
coefficient (the slope of Hill plot) is often used as a
measure of the extent of cooperativity, and a Hill coef-
ficient < 1.0 might suggest negative cooperativity [24].
However, the average Hill coefficient calculated for
PCSK9-WT was equal to unity (0.98 ± 0.06, n = 3),
and that obtained for PCSK9-D374Y was not signifi-
cantly different from unity (0.87 ± 0.07, n = 3) (not
shown).
Kinetic characteristics of [
125
I]TC-PCSK9 binding
to HepG2 cells
To determine whether the kinetics of binding of
PCSK9 to HepG2 cells can be described as a simple
bimolecular reaction, the kinetics of [
125
I]TC-PCSK9
and [
125
I]TC-PCSK9-D374Y dissociation from and
association with HepG2 cells were determined.
Kinetic association
The time course of [
125
I]TC-PCSK9 association with
HepG2 cells at 4 °C is shown in Fig. 3A,B. Specific
binding of [
125
I]TC-PCSK9 (5 lgÆmL
)1
,70nm) to cells
Characterization of the binding of PCSK9 to intact cells S. A. Mousavi et al.
2940 FEBS Journal 278 (2011) 2938–2950 ª 2011 The Authors Journal compilation ª 2011 FEBS
reached an apparent equilibrium within 4 h. Binding
of [
125
I]TC-PCSK9-D374Y (1 lgÆmL
)1
,14nm)to
HepG2 cells indicates that [
125
I]TC-PCSK9-D374Y at
a concentration five times lower than that of [
125
I]TC-
PCSK9 bound to the cells in a similar time-dependent
manner, and approached binding equilibrium at nearly
the same rate, suggesting a higher affinity of [
125
I]TC-
PCSK9-D374Y for cell surface LDLR. For both
[
125
I]TC-PCSK9 and [
125
I]TC-PCSK9-D374Y, the time
courses of binding were biphasic, and data from indi-
vidual association curves were well fitted by a two-
phase exponential association model, suggesting that
surface binding has two components, one rapid and
one slow. The half-time for association of [
125
I]TC-
PCSK9 with the rapid component, representing 35%
(± 5.3%) of specific equilibrium binding, was 6.6 min
(± 1.03 min), whereas the half-time for binding to the
slow component was 94 min (± 23 min) (n = 3). The
corresponding half-times for [
125
I]TC-PCSK9-D374Y
binding were 6.1 min (± 0.8 min) and 89 min
(± 9 min) (n = 3), respectively. The observed associa-
tion rate constants k
obs
for the rapid phase [k
obs(rapid)
]
and for the slow phase [k
obs(slow)
] are shown in
Table 2.
Kinetic dissociation
The data in Fig. 4 show the time course of [
125
I]TC-
PCSK9 and [
125
I]TC-PCSK9-D374Y dissociation from
HepG2 cells. It is evident that the dissociation of both
[
125
I]TC-labeled ligands is biphasic, with two kinetic
components. Data from individual dissociation curves
were best described by a model of two exponential
decay phases. Approximately 25% of the bound
[
125
I]TC-PCSK9 dissociated during the rapid phase,
with a half-time of 19 min (± 2.7 min), and the
remaining bound [
125
I]TC-PCSK9 dissociated slowly,
with a half-time of 270 min (± 22 min) (n = 3). The
A
B
C
Fig. 2. Nonlinear regression and Scatchard analyses of binding-inhi-
bition data. (A) Inhibition of [
125
I]TC-PCSK9 (2.5 lgÆmL
)1
) binding to
HepG2 cells ( 9.8 · 10
5
) by increasing concentrations of unlabeled
wild-type (upper curve), D374Y (lower curve) and S127R (middle
curve) variants of PCSK9. Bars the denote range of duplicate deter-
minations. For wild-type PCSK9 and PCSK9-S127R, only the upper
and lower, respectively, halves of the ranges are shown, to avoid
overlap of the error bars. The results are expressed as percentage
of control (c.p.m. in the absence of unlabeled ligand), plotted
against the concentration of unlabeled ligands. (B) Inhibition of
[
125
I]TC-PCSK9 binding to HepG2 cells (9.2–9.8 · 10
5
) by increasing
concentrations of unlabeled wild-type PCSK9. Error bars are stan-
dard deviations for data from three separate experiments [including
the one shown in (A)]. The results are expressed as a percentage
of c.p.m. in the absence of unlabeled wild-type PCSK9, plotted
against the concentration of unlabeled wild-type PCSK9. Inset: a
representative Scatchard plot of the competition for [
125
I]TC-PCSK9
binding by unlabeled wild-type PCSK9. (C) Inhibition of [
125
I]TC-
PCSK9-D374Y (1 lgÆmL
)1
) binding to HepG2 cells (9.2–9.8 · 10
5
)
by increasing concentrations of unlabeled PCSK9-D374Y. Error bars
are standard deviations for data from three separate experiments.
The results are expressed as a percentage of c.p.m. in the absence
of unlabeled PCSK9-D374Y, plotted against the concentration of
unlabeled PCSK9-D374Y. Inset: a representative Scatchard plot of
the competition for [
125
I]TC-PCSK9-D374Y binding by unlabeled
PCSK9-D374Y. All of the data shown in the three panels were
described best by a two-binding site model. Scatchard plots of the
competition for [
125
I]TC-PCSK9 binding by PCSK9-S127R and
PCSK9-D374Y [shown in (A)] exhibit curvatures similar to those of
wild-type PCSK9 and PCSK9-D374Y (not shown). Each curve was
analyzed separately, and the parameters (IC
50
, K
d
) determined from
these experiments (means ± standard deviations) are shown in
Table 1. The asterisk indicates the range of duplicate determina-
tions from a single experiment.
S. A. Mousavi et al. Characterization of the binding of PCSK9 to intact cells
FEBS Journal 278 (2011) 2938–2950 ª 2011 The Authors Journal compilation ª 2011 FEBS 2941
amount of [
125
I]TC-PCSK9 that remained bound after
6 h was about 40%. Approximately 20% of the bound
[
125
I]TC-PCSK9-D374Y dissociated rapidly, with a
half-time of 21 min (± 3.7 min), and the remaining
bound [
125
I]TC-PCSK9-D374Y dissociated more
slowly, with a half-time of 297 min (± 25 min)
(n = 3). The fraction of [
125
I]TC-PCSK9-D374Y that
remained bound after 6 h was about 45%. The dissoci-
ation rate constants [k
off
] for the rapid phase [k
off(rapid)
]
and for the slow phase [k
off(slow)
] are shown in Table 2.
Taken together with the association data, these
results suggest that the increased affinity of PCSK9-
D374Y for cell surface LDLR is mainly determined by
the rate of association. It should be noted that quanti-
tative analysis of the more rapid phase of association
requires measurement of the binding on time scales of
minutes, a time resolution that is difficult to achieve in
experiments with adherent cell cultures. This phase
appears to be complete by the second measurable time
point (15 min) in our system, and this precludes esti-
mation of the association rate constant [k
on
] and the
kinetic K
d
(i.e. the K
d
representing the k
off
⁄ k
on
ratio).
Dissociation of [
125
I]TC-PCSK9 in the presence of
unlabeled PCSK9
In order to establish whether the [
125
I]TC-PCSK9 bound
to the cells after 6 h was dissociable, dissociation experi-
ments were performed in the absence and presence of
unlabeled PCSK9 (Fig. 4A). In the presence of a high
concentration of unlabeled wild-type PCSK9, the half-
time of the rapid phase was reduced from 19 to 16 min
and the half-time of the slow phase was reduced from
270 to 154 min. In the presence of unlabeled PCSK9-
D374Y, the half-time of the rapid phase of [
125
I]TC-
PCSK9-D374Y dissociation was reduced from 21 to
19 min, and the half-time of the slow phase was reduced
from 297 to 179 min (Fig. 4B). These results suggest
that the reason for ligand remaining bound to cells after
6 h is not irreversibility of the binding.
Dissociation of [
125
I]TC-PCSK9 at low pH
As the affinity of PCSK9 for the ECD or the EGF-A
domain of LDLR is known to increase at acidic pH
Table 1. Parameters obtained from binding-inhibition experiments. Best-fit values for IC
50
were derived from nonlinear regression analysis.
K
d
values were derived separately from Scatchard plots. High and low represent affinities of binding sites for unlabeled ligands. Values in
parentheses indicate the number of experiments performed. The data from each experiment were analyzed separately, and mean val-
ues ± standard deviations were calculated from these values.
Labeled PCSK9 Competitive ligand
IC
50
(nM) K
d
(nM)
High Low High Low
[
125
I]TC-PCSK9 PCSK9-wild type (3) 374 ± 82 2500 ± 400 626 ± 113 2800 ± 300
[
125
I]TC-PCSK9 PCSK9-S127R (1) 263 1900 548 2900
[
125
I]TC-PCSK9 PCSK9-D374Y (1) 68.5 1540 107 1250
[
125
I]TC-PCSK9-D374Y PCSK9-D374Y (3) 92 ± 17 1200 ± 300 125 ± 20 1600 ± 200
Fig. 3. Association time courses of the binding of [
125
I]TC-PCSK9
and [
125
I]TC-PCSK9-D374Y to HepG2 cells at 4 °C. Cells ( 9.2–
9.5 · 10
5
) were incubated for the indicated times in binding
medium containing [
125
I]TC-PCSK9 (5 lgÆmL
)1
)or[
125
I]TC-PCSK9-
D374Y (1 lgÆmL
)1
). At each time point, the cells were washed, and
the specific binding was determined. (A) Binding presented as per-
centage of total radioactivity added. (B) Binding presented as the
amount of [
125
I]TC-labeled ligand specifically bound. Error bars are
standard deviations for data from three separate experiments. The
binding data were normalized for cell number (per 10
6
cells). The
curves were fitted with the two-phase exponential association
model.
Characterization of the binding of PCSK9 to intact cells S. A. Mousavi et al.
2942 FEBS Journal 278 (2011) 2938–2950 ª 2011 The Authors Journal compilation ª 2011 FEBS
[15,25], it is believed that, subsequent to internalization
of the PCSK9–LDLR complex, LDLR is diverted to a
degradation pathway, owing to persistence of the com-
plex at endosomal pH [15]. However, the rates of dis-
sociation of PCSK9 that has previously bound to
LDLR at neutral pH have not been determined under
different pH conditions to test this hypothesis. The
effect of pH on the dissociation of [
125
I]TC-PCSK9
was measured at pH 6.2 (to mimic the early endosomal
pH). As shown in Fig. 4A, low pH did indeed mark-
edly reduce the dissociation of [
125
I]TC-PCSK9 from
cells. Dissociation occurred as a monophasic process
with a rate constant [k
off
] of 0.0009 ± 0.00012 min
)1
(n = 3), which corresponds to a half-time of dissocia-
tion of 770 min. Lowering the pH of dissociation med-
ium to 6.2 also led to monophasic and slow
dissociation of [
125
I]TC-PCSK9-D374Y with a half-
time of 810 min (Fig. 4B).
This observation is in marked contrast to what is
seen in many receptor systems, including the insulin
receptor [26], where lowering of the pH leads to disso-
ciation of ligand from receptor. The reduced dissocia-
tion at low pH may reflect dissociation from a single
high-affinity site or, alternatively, it may reflect the
sum of two dissociation processes. It is likely that a
similar mechanism may be at work in the slightly
acidic early endosomal compartments, where reduced
pH will decrease dissociation of the internalized
PCSK9–LDLR complexes.
Effect of association time on the dissociation of
[
125
I]TC-PCSK9
The dependence of dissociation of [
125
I]TC-PCSK9
and [
125
I]TC-PCSK9-D374Y on the length of associa-
tion time was investigated to determine whether the
proportions of the two kinetic components correspond
to the presence of two distinct receptor sites ⁄ states
with different and fixed affinities. The prediction of
this mechanism is that the proportions of the two
components will be constant ( 3 : 1 for [
125
I]TC-
PCSK9 and  4 : 1 for [
125
I]TC-PCSK9-D374Y) and
independent of the length of association time. This
prediction was tested by comparing the dissociation
rate for binding under the pre-equilibrium conditions
(60 min) and equilibrium conditions (240 min). As can
be seen in Fig. 5A, dissociation of [
125
I]TC-PCSK9
was biphasic at both association times. Approximately
42% and 22% of dissociation occurred in the rapid
phase after short and long incubation times, respec-
tively. The dependence of the kinetics of [
125
I]TC-
PCSK9-D374Y dissociation on the length of binding
time was also examined. Again, dissociation from the
rapid component was found to be faster at 60 min
than at 240 min (36% versus 19% under equilibrium
conditions) (Fig. 5B).
These data suggest that there is a fraction of rapidly
dissociating receptors,  40% after 60 min of associa-
tion, that converts with time to a receptor state that
releases bound ligand very slowly. The size of the frac-
tion undergoing conversion may be larger at shorter
association times (i.e. shorter than 60 min). However,
determination of the half-time for this conversion
requires an analysis of shorter-term aspects of this pro-
cess, which is not possible in our system, owing to the
relatively long durations of such experiments.
Discussion
This is the first study aimed at characterizing the bind-
ing of PCSK9 to intact cells by using radiolabeled
PCSK9. Several observations indicate that LDLR is
the main surface receptor mediating PCSK9 binding to
HepG2 cells. First, the number of LDLRs on HepG2
cells increased following growth in the absence of lipo-
proteins, and there was a corresponding increase in the
binding of [
125
I]TC-PCSK9 and [
125
I]TC-PCSK9-
D374Y. Second, the extent of specific binding of both
[
125
I]TC-labeled ligands was a linear function of the
cell density. Finally, the binding is specific, as the
Table 2. Parameters obtained from kinetic experiments. k
off
values were obtained by fitting the time course data with a two-exponential
decay phase model. k
off(rapid)
and k
off(slow)
are the dissociation rate constants for the rapid and slow dissociation components, respectively.
k
obs
values were obtained by fitting the time course data with a two-exponential association model. k
obs(rapid)
and k
obs(slow)
are the observed
association rate constants for the rapid and slow association phases, respectively. The values for the constants are means ± standard devia-
tions of three experiments. The data from each experiment were analyzed separately. The concentrations used are those described in the
text.
Labeled ligand
k
off
(min
)1
) k
obs
(min
)1
)
k
off(rapid)
k
off(slow)
k
obs(rapid)
k
obs(slow)
[
125
I]TC-PCSK9 0.036 ± 0.005 0.0026 ± 0.0002 0.108 ± 0.016 0.0077 ± 0.0020
[
125
I]TC-PCSK9-D374Y 0.033 ± 0.006 0.0023 ± 0.0002 0.116 ± 0.017 0.0079 ± 0.0008
S. A. Mousavi et al. Characterization of the binding of PCSK9 to intact cells
FEBS Journal 278 (2011) 2938–2950 ª 2011 The Authors Journal compilation ª 2011 FEBS 2943
binding of [
125
I]TC-PCSK9 was reduced by unlabeled
wild-type as well as by two mutant variants of PCSK9
and LDL, but not by an unrelated ligand, suggesting
that they all compete with [
125
I]TC-PCSK9 for the
same receptor.
Linear [27–29] and curvilinear [30,31] Scatchard
plots for LDL binding to LDLR are observed. Analy-
sis of inhibition curves of [
125
I]TC-PCSK9 and
[
125
I]TC-PCSK9-D374Y by the Scatchard method
consistently showed curvilinear plots that implicated
the presence of high-affinity and low-affinity sites ⁄ -
states with affinities for PCSK9 that differ approxi-
mately five-fold. An apparently good fit of nonlinear
regression analysis of binding data to a two-site model
was also obtained, suggesting that the equilibrium
binding of PCSK9 to cell surface LDLR is not a sim-
ple bimolecular reaction (see Doc. S1, model A).
One possibility that could explain the observed cur-
vilinear Scatchard plots is that unlabeled and labeled
ligands have different affinities for the receptors [32].
However, this seems to be less likely, as the presence
of two classes of PCSK9 binding site were also
observed in kinetic experiments where only [
125
I]TC-
labeled ligands were employed. Moreover, we believe
that the [
125
I]TC-labeling method, in contrast to the
direct
125
I-labeling method, does not appreciably alter
the binding properties of PCSK9, as [
125
I]TC-PCSK9-
Fig. 5. Dissociation kinetics of [
125
I]TC-PCSK9 (A) and [
125
I]TC-
PCSK9-D374Y (B) as a function of time of association: HepG2 cells
( 9 · 10
5
) were incubated at 4 °C in medium containing [
125
I]TC-
labeled ligand for 60 or 240 min. After removal of unbound [
125
I]TC-
labeled ligand, the cells were incubated at 4 °C in fresh medium,
and dissociation was measured as described in Experimental proce-
dures. Data are presented as percentage of total ligand bound at
zero time (100%). The 240-min data are from a single experiment,
and error bars represent range of duplicate determinations. Error
bars in 60-min curves represent mean ± one-half the range from
two independent experiments, each performed in duplicate.
Fig. 4. Dissociation time courses and effects of unlabeled ligands
and low pH on the dissociation rates of [
125
I]TC-PCSK9 (A) and
[
125
I]TC-PCSK9-D374Y (B) from HepG2 cells. Binding to equilibrium
and removal of unbound [
125
I]TC-labeled ligands were performed as
described in Experimental procedures. The cells were then incu-
bated in dissociation medium (pH 7.4) without (control) or with
unlabeled ligand or medium (pH 6.2), and dissociation of specifically
bound ligands was followed as a function of time. Data are pre-
sented as percentage of total ligand bound at zero time (100%).
Error bars are standard deviations for data from three separate
experiments. Asterisks indicate error bars representing mean ±
one-half the range from two separate experiments. Dissociation in
the absence (control) and presence of unlabeled ligand was best
described by a two-exponential decay phase model, whereas disso-
ciation at low pH was best described by a one-exponential decay
phase model. Final concentrations of unlabeled ligands in the disso-
ciation medium were 120 lgÆmL
)1
(wild-type PCSK9) and
30 lgÆmL
)1
(PCSK9-D374Y). If the K
d
values estimated here are
assumed, then about 70% of the high-affinity sites and about 12%
of the low-affinity sites are expected to be occupied at the concen-
trations of unlabeled ligands used.
Characterization of the binding of PCSK9 to intact cells S. A. Mousavi et al.
2944 FEBS Journal 278 (2011) 2938–2950 ª 2011 The Authors Journal compilation ª 2011 FEBS
D374Y consistently displayed a much higher affinity
for HepG2 cell surface receptors than did [
125
I]TC-
PCSK9. Binding to sites other than LDLR, such as
LDLR related protein 1 (LRP1) [4], may also be
responsible for the observed curvature. However, we
consider this possibility to be less likely, because LRP1
has been shown to be not regulated by cellular choles-
terol levels [33], and therefore cannot account for the
observed increased binding of [
125
I]TC-PCSK9 to
HepG2 cells grown in the absence of lipoproteins. It is
more likely that the observed heterogeneity in binding
of [
125
I]TC-PCSK9 to HepG2 cells is attributable to
binding to different populations of LDLR that exist
prior to ligand binding (see below).
Previous Biacore studies have primarily used the
ECD of LDLR to determine the affinity and kinetics
of binding of PCSK9. The K
d
values reported for wild-
type PCSK9 interaction with the ECD of LDLR at
neutral pH [19,25,34,35] differ by about an order of
magnitude (ranging from 90 to 840 nm). The K
d
of
wild-type PCSK9 for the high-affinity LDLRs
(626 ± 113 nm) in intact cells estimated from Scat-
chard plots is within this range. The calculated K
d
value for PCSK9-D374Y binding to the high-affinity
sites was 125 ± 20 nm. Other investigators have
reported an apparent K
d
for binding of PCSK9-D374Y
to the ECD of LDLR that is similar to (101 nm) [19]
or 20-fold lower (6 nm) [25] than the K
d
estimated
here. The apparent K
d
of PCSK9-S127R binding to
the high-affinity LDLR site (548 nm), as measured by
its ability to inhibit [
125
I]TC-PCSK9 binding to HepG2
cells, was slightly lower (i.e. slightly higher affinity)
than that for wild-type PCSK9, and is comparable to
the 648 nm K
d
obtained in a Biacore study [19]. The
K
d
values derived for the lower-affinity class of sites
are shown in Table 1. In this context, it is worth men-
tioning that the existence of high-affinity (32 nm) and
low-affinity (86 nm) states has previously been demon-
strated for binding of PCSK9-S127R to the ECD of
LDLR at pH 7.5 [25]. This study also found high-
affinity (1 nm) and low-affinity (42 nm) binding states
for the interaction between wild-type PCSK9 and the
ECD of LDLR at pH 5.4, whereas PCSK9-D374Y
binds with only one affinity (K
d
=6nm) at pH 7.5
and with a slightly higher affinity (K
d
= 1.6 nm)at
pH 5.4. It therefore seems likely that the ECD of
LDLR also adopts different conformations when
immobilized on the biosensor chip.
The observation of two kinetic components in the
association and dissociation kinetics also suggests the
presence of two populations of binding site. However,
in experiments that examined the dissociation as a
function of association time (Fig. 5), it was found that
increasing the association time increased the propor-
tion of the slowly dissociating component, at the
expense of the component with rapid dissociation. This
result cannot be simply explained by the presence of
two populations of LDLR that have different and
fixed affinities for PCSK9 (see Doc. S1, model B). A
possible explanation might be that binding of PCSK9
to the low-affinity form of LDLRs is followed by a
slow conformational change of the ligand–receptor
complex to the higher-affinity state, whereas this con-
formational transition is faster when PCSK9 binds to
the high-affinity form of receptor.
A model (see Doc. S1, model C) that appears to be
consistent with the kinetic data is one in which
LDLRs on HepG2 cells are in equilibrium between
monomer and dimer states and PCSK9 interacts with
both populations of the receptor via a two-step reac-
tion in which the first binding step, representing bind-
ing to the EGF-A domain, is followed by binding of
PCSK9 to a second site within the receptor. In this
model, dimeric and monomeric receptor states bind
PCSK9 with equal affinity, but they differ in their con-
version rates, i.e. rate constants governing the confor-
mational change that leads to the second binding step.
Thus, the rapid phase of PCSK9 association could rep-
resent binding of PCSK9 to dimeric receptors that
release bound ligand slowly because they convert rap-
idly. The slow phase of association could represent
binding to monomeric receptors that release bound
ligand rapidly because they convert slowly. The pro-
posed model is supported by the finding that a signifi-
cant proportion of LDLRs in the plasma membrane
pre-exist as noncovalent dimers (or higher oligomers)
in coated pits [36–38] or even outside coated pits [39],
and by the recent demonstration that PCSK9 can also
bind, via its C-terminal domain, to the LDL-binding
domain of LDLR [18].
The molecular basis of the enhanced rate of dissoci-
ation observed in the presence of unlabeled ligand is
unclear. This phenomenon has often, but not always,
been interpreted as indicative of the presence of nega-
tive cooperativity, i.e. a decrease in affinity with
increasing site occupancy [40]. At the present time, a
mechanistic explanation of negative cooperativity, if
present, in this system would be difficult, although
negative cooperativity among partially occupied
dimeric receptors or between two binding sites on a
monomeric divalent receptor [41] cannot be excluded.
It should be noted that, given the small amount of
[
125
I]TC-PCSK9 initially bound and its low affinity
for LDLR, rebinding of dissociated [
125
I]TC-PCSK9
from the bulk solution cannot account for the slowly
dissociating component, although rebinding from the
S. A. Mousavi et al. Characterization of the binding of PCSK9 to intact cells
FEBS Journal 278 (2011) 2938–2950 ª 2011 The Authors Journal compilation ª 2011 FEBS 2945
putative ‘unstirred layer’ surrounding the cells [42] can-
not be excluded.
The presence of two apparent classes of binding site
with different affinities for PCSK9 on HepG2 cells
raises the question of whether both classes of site are
involved in internalization and whether the rate con-
stants of association and dissociation for [
125
I]TC-
PCSK9 at 37 °C are similar to values obtained at
4 °C. Our preliminary data indicate that HepG2 cells
are able to internalize and degrade [
125
I]TC-PCSK9 at
37 °C. However, the interaction of ligands with cell
surface receptors at 37 °C is a function not only of the
rate constants of association and dissociation, but also
of the endocytic rate constant, and measurements of
these rate constants require a method to discriminate
between the surface-bound and internalized ligand [43].
However, an accurate measurement of
125
I-TC-PCSK9
association and dissociation rates at 37 °C is difficult
to obtain, because, as discussed in Results, in contrast
to many ligand–receptor systems, acid wash does not
favor dissociation of [
125
I]TC-PCSK9 from the plasma
membrane. We are currently trying to develop a wash
method that can effectively remove cell surface-bound
[
125
I]TC-PCSK9.
Experimental procedures
Materials
Culture media and antibiotics, l-glutamine and nonessential
amino acids were from Gibco BRL (Invitrogen, Carlsbad,
CA, USA). Antibody against LDLR was from RDI
Research Diagnostic (Concord, MA, USA). BSA and fetal
bovine serum were from Sigma Aldrich (St Louis, MO,
USA). Na
125
I was purchased from PerkinElmer (Waltham,
MA, USA). IodoGen-precoated tubes were from Pierce
Biotechnology (Rockford, IL, USA). All other chemicals
and reagents were obtained from Sigma Aldrich unless
otherwise specified.
Protein expression and purification
PCSK9-D374Y and PCSK9-S127R are two naturally occur-
ring gain-of-function mutants of PCSK9 that cause severe
hypercholesterolemia [1,11,12]. Histidine-tagged PCSK9s
(wild type, D374Y, and S127R) were produced by transfec-
tion of HEK239 cells, and purified from conditioned media
as previously described [44].
Radiolabeling of proteins
We initially attempted to investigate the binding of PCSK9
to HepG2 cells using PCSK9 directly labeled with
125
I
([
125
I]PCSK9), which reacts with tyrosines of the protein.
However, analysis in single-point binding assays showed
only 50–60% of the cell-associated [
125
I]PCSK9 could be
inhibited in the presence of a large excess of unlabeled
PCSK9, indicating a high level of nonspecific binding. This,
combined with the low affinity of PCSK9 for LDLR, made
reliable measurements of specific binding difficult. To over-
come the problem of low specific binding, we used labeling
by [
125
I]TC, which reacts with lysines, and we found
[
125
I]TC labeling of PCSK9 to be more suitable for equilib-
rium and kinetic binding studies, because of the much
lower level of nonspecific binding. The reason why different
labeling methods produce molecules with different binding
properties is unclear, but the results suggest that the bind-
ing of [
125
I]PCSK9 to a nonreceptor site is particularly
enhanced by the radio-iodination of a tyrosine(s). It should
be noted that the EGF-A-binding region of PCSK9 con-
tains no tyrosines (or lysines).
Purified wild-type PCSK9 was covalently coupled to
[
125
I]TC by the method of Pittman et al. [45], with modifi-
cations as described previously [46]. Briefly, [
125
I]TC was
prepared by reacting TC (6 lLof10mm solution in
NaCl ⁄ P
i
) with Na
125
I (1.0 mCi) in IodoGen-precoated
tubes (Pierce) for 30–40 min at room temperature, followed
by transfer to a tube containing cyanuric chloride (6 lLof
10 mM solution in acetonitrile) and potassium iodide (6 lL
of 0.1 m solution) for 3 min. The activated [
125
I]TC adduct
was then incubated with wild-type PCSK9 (300–400 lgin
200 lL of carbonate buffer containing 0.5 mm CaCl
2
,
pH 8.9) for 30–45 min. Unreacted [
125
I]TC (and free
125
I)
were removed by gel filtration with Sephadex G25 columns
(PD-10; GE Healthcare) equilibrated with NaCl ⁄ P
i
contain-
ing 0.2 mm CaCl
2
.[
125
I]TC labeling of PCSK9-D374Y was
carried out as previously described, except that 80–100 lg
of purified protein was used for labeling. Specific activities
obtained were in the range of 6–7 · 10
5
c.p.m. lg
)1
. The
quality and integrity of the [
125
I]TC-labeled proteins were
evaluated by SDS ⁄ PAGE followed by EZBlue staining
(Pierce) and autoradiography of the gel. [
125
I]TC-PCSK9 is
stable, and can be stored at 4 °C for at least 2 weeks. For
direct iodination, purified PCSK9 (100 lg in 100 lLof
NaCl ⁄ P
i
containing 0.5 mm CaCl
2
, pH 7.4) was incubated
with Na
125
I (0.3 mCi) in IodoGen-precoated tubes for 10–
15 min at room temperature. The reaction was stopped by
transfer to a tube containing 0.9 mL of NaCl ⁄ P
i
containing
0.5 mm CaCl
2
(pH 7.4). Free
125
I was removed by gel filtra-
tion as described above. Goat anti-(rabbit IgG) was radio-
labeled with Na
125
I as described for direct iodination of
PCSK9, except that CaCl
2
was omitted.
Cell culture, buffers, and cell treatments
HepG2 cells (European Collection of Cell Cultures, Porton
Down, UK) were routinely cultured in collagen-coated
75-cm
2
tissue culture flasks (BD Biosciences, San Diego,
Characterization of the binding of PCSK9 to intact cells S. A. Mousavi et al.
2946 FEBS Journal 278 (2011) 2938–2950 ª 2011 The Authors Journal compilation ª 2011 FEBS
CA, USA) in MEM supplemented with 200 mml-gluta-
mine, nonessential amino acids, and 10% fetal bovine
serum (referred to as complete growth medium) in a
5% CO
2
atmosphere at 37 °C. For binding and kinetic
experiments, cells were plated at 250 000–320 000 cells per
well in collagen-coated 12-well culture plates (BD Bio-
sciences, San Diego, CA, USA). After incubation for 22–
24 h at 37 °C in complete growth medium, cells were
washed twice with 1 mL of Opti-MEM and incubated in
Opti-MEM containing 3% lipoprotein-depleted serum
(LPDS) for 24–26 h at 37 °C to upregulate LDLR. In some
experiments, the cells were incubated in Opti-MEM without
LPDS. Cell-free wells (blanks) were treated in an identical
manner, and were included in all experiments. Collagen-
coated culture plates were used to avoid cell detachment
during the relatively long (> 13 h) durations of some
experiments. All experiments were performed at 4 °Cto
prevent endocytosis of bound ligand. The cells were also
kept at 4 °C during the washing steps. The standard bind-
ing medium for all experiments was DMEM containing
20 mm Hepes and 1% BSA (pH 7.4). The dissociation
(chase) medium was the same as that for binding, except
that, in some experiments, the pH of the chase medium was
adjusted to 6.2 with Mes at 4 °C. Exposure of cells to low
pH did not damage the cells during the chase period
( 6 h), as judged by the ability of cells to exclude Trypan
blue dye. At the end of each experiment, two wells were
treated with trypsin and the average cell number was deter-
mined with a hemocytometer. All experiments were per-
formed on several different HepG2 cell batches and, with
the exception of PCSK9-S127R (one preparation), at least
three different wild-type PCSK9 and PCSK9-D374Y prepa-
rations and five different preparations of [
125
I]TC-labeled
proteins were used.
Determination of specific binding of
[
125
I]TC-PCSK9 to HepG2 cells
Varying numbers of HepG2 cells (70 000, 140 000 and
280 000) were seeded in 12-well plates and incubated either
for 48 h in complete growth medium or for 24 h in com-
plete growth medium, and then for 26 h in 3% LPDS-con-
taining Opti-MEM. In experiments with cells grown in
complete growth medium for 48 h, cells were incubated in
lipoprotein-free medium for 2 h at 37 °C before the start of
the experiments, to allow internalization of cell surface-
bound LDL. After 50 h of incubation at 37 °C, cells were
washed once with 1 mL of DMEM and incubated in the
same medium for 15 min at 4 °C. The medium was then
removed, and triplicate wells were incubated with 0.5 mL
of incubation medium containing 10 lgÆmL
)1
of antibody
against LDLR for 90 min at 4 °C. The cells were then
washed, and the amount of bound antibody against LDLR
was measured by subsequent binding of
125
I-labeled goat
anti-(rabbit IgG) (10 lgÆmL
)1
). A control in which the anti-
body against LDLR was omitted was used to measure the
amount of nonspecific binding. Triplicate wells were also
incubated with 0.5 mL of incubation medium containing
either [
125
I]TC-PCSK9 (5 lgÆmL) or [
125
I]TC-PCSK9-
D374Y (1 l g Æ mL
)1
) for 4 h at 4 °C. The cells were washed
once with 1 mL of cold wash buffer, NaCl ⁄ P
i
containing
0.1 mm CaCl
2
and 0.5% BSA, and three times with 1 mL
of wash buffer without BSA. The cells were solubilized in
0.5 mL of 0.2 m NaOH, and transferred to counting tubes
following a 15-min incubation at room temperature. Wells
were washed with an additional 0.5 mL of 0.2 m NaOH,
added to the counting tube, and counted on a gamma
counter. Similar experiments were carried out in single-
point binding assays at high initial cell density.
Binding-inhibition experiments
Cells grown in 12-well plates were washed once with 1 mL
of DMEM and incubated in the same medium for at least
15 min at 4 °C. The medium was removed, and cells were
incubated with increasing concentrations of unlabeled (seri-
ally diluted two-fold) proteins in a total volume of 480 lL
at 4 °C for 15 min. Labeled ligand was added in a small
volume (20 lL) and the cells were incubated at 4 °C for
4 h. At the end of incubation, the cells were washed once
with 1 mL of cold wash buffer and three times with 1 mL
of wash buffer without BSA. The wash procedure took
 4 min per plate. After washing, cells were solubilized and
radioactivity was measured as described above.
Kinetic association experiments
Cells grown in 12-well plates were washed as described
above. The medium was removed, and the cells were incu-
bated with 0.5 mL of binding medium containing [
125
I]TC-
PCSK9 ( 5 lgÆmL
)1
,70nm)or[
125
I]TC-PCSK9-D374Y
( 1 lgÆmL
)1
,14nm). At various times, cells were washed
four times and solubilized, and radioactivity was measured
as previously described. A 200-fold excess of unlabeled
PCSK9-D374Y was added to selected wells, in order to
allow estimation of nonspecific binding. Specific binding
was calculated by subtracting nonspecific from total
binding.
Kinetic dissociation experiments
Cells grown in 12-well plates were washed as described
above, and were incubated for 4 h at 4 °C in binding med-
ium containing [
125
I]TC-PCSK9 ( 5 lgÆmL
)1
)or[
125
I]TC-
PCSK9-D374Y ( 1 lgÆ mL
)1
). At the end of incubation,
the cells were washed four times with wash buffer as
described above, and then incubated in fresh binding
medium. At various times, cells were washed once with
1 mL of cold DMEM and solubilized, and radioactivity was
measured as described above. Nonspecific binding was
S. A. Mousavi et al. Characterization of the binding of PCSK9 to intact cells
FEBS Journal 278 (2011) 2938–2950 ª 2011 The Authors Journal compilation ª 2011 FEBS 2947
measured in two parallel wells in the presence of an excess of
unlabeled PCSK9-D374Y. Radioactivity released from cells
following 6 h of dissociation was more than 90% precipita-
ble in 10% trichloroacetic acid, indicating that the majority
of bound [
125
I-TC-PCSK9 (and [
125
I]TC-PCSK9-D374Y)
molecules remained intact and were still on the cell surface.
Data analysis
All binding and kinetic data were fitted by nonlinear regres-
sion with prism 5 (GraphPad Software, CA, USA). For
inhibition-binding curves, the raw data were analyzed
according to one-site-fit and two-site-fit log
IC50
models. The
data were also analyzed according to the Scatchard method
[47]. Linear regression analyses of binding data gave dissoci-
ation constants (K
d
), calculated from the reciprocal of the
slopes. Association data were fitted by either the one-phase
exponential association equation Y = Y
0
+ (plateau )
Y
0
)*[1 ) exp() Kx)] or the two-phase exponential associa-
tion equation Y = Y
0
+ SpanFast*[1 ) exp() KFast*
X)] + SpanSlow*[1 ) exp() K
Slow
X)]. Dissociation data were
fitted by either the one-phase exponential decay equation
Y =(Y
0
) plateau)*exp() KX) + plateau or the two-phase
exponential decay equation Y = plateau + Span-
Fast*exp() K
Fast
X) + SpanSlow*exp() K
Slow
X). The data
were also analyzed by plotting dissociation data according
to ln(B
t
⁄ B
0
) versus time, where B
0
is binding at the onset of
dissociation, and B
t
is the binding remaining at time t. Better
fits were determined with the F-test. Differences were consid-
ered to be significant when P was < 0.05.
Acknowledgements
We thank G. Griffiths (Department of Molecular Bio-
sciences, University of Oslo) for critical reading of the
manuscript.
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Supporting information
The following supplementary material is available:
Doc. S1. Models.
This supplementary material can be found in the
online version of this article.
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Characterization of the binding of PCSK9 to intact cells S. A. Mousavi et al.
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