A Ligand-induced Conformational Change in Apolipoprotein(a)
Enhances Covalent Lp(a) Formation*
Lev
Becker,
Bradley A.
Webb,
Seth
Chitayat,
Michael E.
Nesheim, and
Marlys L.
Koschinsky
From the Department of Biochemistry, Queen's University,
Kingston, Ontario K7L 3N6, Canada
Received for publication, December 17, 2002, and in revised form, January 31, 2003
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ABSTRACT |
Lipoprotein(a) (Lp(a)) assembly proceeds via a
two-step mechanism in which initial non-covalent interactions between
apolipoprotein(a) (apo(a)) and low density lipoprotein precede
disulfide bond formation. In this study, we used analytical
ultracentrifugation, differential scanning calorimetry, and intrinsic
fluorescence to demonstrate that in the presence of the lysine analog
-aminocaproic acid, apo(a) undergoes a substantial conformational
change from a "closed" to an "open" structure that is
characterized by an increase in the hydrodynamic radius (~10%), an
alteration in domain stability, as well as a decrease in tryptophan
fluorescence. Although
-aminocaproic acid is a well characterized
inhibitor of the non-covalent interaction between apo(a) and low
density lipoprotein, we report the novel observation that this ligand
at low concentrations (100 µM-1 mM) significantly enhances covalent Lp(a) assembly by altering the conformation of apo(a). We developed a model for the kinetics of Lp(a)
assembly that incorporates the conformational change as a determinant
of the efficiency of the process; this model quantitatively explains
our experimental observations. Interestingly, an analogous
conformational change has been previously described for plasminogen
resulting in an increase in the hydrodynamic radius, an increase in
tryptophan fluorescence, and an acceleration of the rate of plasminogen
activation. Although the functions of apo(a) and plasminogen have
diverged considerably, elements of structural and conformational
homology have been retained leading to similar regulation of two
unrelated biological processes.
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INTRODUCTION |
Lipoprotein(a)
(Lp(a))1
is a plasma lipoprotein that has been identified as a risk factor for
the development of a variety of atherogenic disorders including
coronary heart disease (1). Lp(a) is comprised of a low density
lipoprotein (LDL)-like domain that is covalently attached to a unique
glycoprotein, termed apolipoprotein(a) (apo(a)). Lp(a) is similar to
LDL both in lipid composition and in the presence of
apolipoproteinB-100 (apoB-100). As such, the presence of apo(a) likely
confers the unique functional and structural properties attributable to
Lp(a). Lp(a) is assembled extracellularly (2, 3); there is evidence to
suggest that assembly may occur on the hepatocyte surface (4). It is
well known that Lp(a) assembly occurs by a two-step process in which
non-covalent interactions between apo(a) and apoB-100 precede specific
disulfide bond formation (5, 6).
Apo(a) bears a strong primary sequence homology to the serine protease
zymogen plasminogen (7). Plasminogen consists of an N-terminal
"tail" domain, followed by five distinct kringle domains followed
by a trypsin-like latent protease domain. Apo(a) contains multiple
repeats of sequences that resemble plasminogen kringle IV, followed by
sequences that are homologous to the kringle V and protease domains of
plasminogen. The kringle IV domains of apo(a) are further classified
into 10 distinct subclasses; the kringle IV type 2 domain
(KIV2) is present in variable copy number, which forms the
basis for the observed isoform size heterogeneity of apo(a), while the
other nine kringle IV types are each present in single copy (8). The
differences in biological function between apo(a) and plasminogen are a
function both of the very different domain composition of the two
proteins as well as more subtle sequence differences between individual
domains. Individual kringle domains in apo(a), as well as the
protease-like domain, display striking sequence similarity to the
analogous domains in plasminogen (up to 90%). For numerous reasons,
the protease domain in apo(a) cannot be activated by activators of
plasminogen (9, 10), resulting in a protein that lacks fibrinolytic
properties. KIV types 5-8 (KIV5-8) harbor "weak"
lysine-binding sites (11) that display a high affinity for lysine
residues within the N terminus of apoB-100 (12) and that are thought to
orient apo(a) and apoB-100 in an appropriate manner to facilitate
disulfide bond formation. As such, it is not surprising that lysine and lysine analogs such as
-aminocaproic acid (
-ACA) are effective inhibitors of Lp(a) assembly through inhibition of the non-covalent association between apo(a) and apoB-100 (12, 13). Kringle IV type 9 (KIV9) contains an unpaired cysteine residue that mediates disulfide bond formation with apoB-100 to form covalent Lp(a) particles
(2, 3). Apo(a) also contains a "strong" lysine-binding pocket
within kringle IV type 10 (KIV10), that does not contribute to binding to LDL, but rather may mediate binding of apo(a) and Lp(a)
to other physiological ligands such as fibrin (14, 15).
The structure of Lp(a) has been investigated using a variety of
biophysical techniques. Electron microscopy studies indicate that Lp(a)
is a spherical particle in which apo(a) is in close association with
LDL (16), a result that is consistent with both the covalent and
non-covalent interactions that characterize the process of Lp(a)
assembly. Upon the addition of
-ACA, however, Lp(a) undergoes a
dramatic conformational change in which the non-covalent interactions
between apo(a) and apoB-100 are disrupted and the apo(a) component
dissociates away from the LDL particle; the dissociation is not
complete as apo(a) remains anchored to LDL through the disulfide
linkage with apoB-100. This change in conformation has been visualized
as a decrease in the sedimentation coefficient in the analytical
ultracentrifuge (17) and in the appearance of long extended tails on
the Lp(a) particles when viewed under the electron microscope (16). The
conformational change observed in these studies, however, results from
a disruption of non-covalent interactions between apo(a) and apoB-100
in the presence of
-ACA and thus represents an intermolecular
structural change rather than an intramolecular conformational change
in apo(a). Such an intramolecular conformational switch has been previously described for Glu-plasminogen, resulting in a transition from a "closed" to "open" conformation (18). Given the
structural homology between apo(a) and plasminogen, we performed a
thorough examination of the effect of
-ACA on the structure of
apo(a). Fluorescence spectroscopy, analytical ultracentrifugation, and differential scanning calorimetry were used to determine whether apo(a)
undergoes a
-ACA-induced conformational change, while the
significance of this conformational change was assessed with respect to
non-covalent binding between apo(a) and LDL as well as covalent Lp(a) assembly.
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EXPERIMENTAL PROCEDURES |
Purification of Apo(a)--
A 17-kringle (17K)-containing form
of recombinant apo(a) (r-apo(a)) as well as a construct encoding the
weak lysine-binding kringles present within apo(a) KIV5-8
were constructed and expressed as previously described (19, 20). Both
r-apo(a) derivatives were purified from the conditioned media (CM) of a stably expressing human embryonic kidney (HEK) 293 cell line (19) by
lysine-Sepharose (Amersham Biosciences) affinity chromatography (13).
Measurements of Intrinsic Fluorescence--
Intrinsic
fluorescence measurements of r-apo(a) were performed using an LS50B
Luminescence Spectrometer (PerkinElmer Life Sciences). 17K or
KIV5-8 r-apo(a) (70 nM) was titrated with a
range of
-ACA concentrations. Titrations were performed in HEPES-buffered saline (HBS; 20 mM HEPES, pH 7.4; 150 mM NaCl) containing 0.01% Tween 20 in a quartz cuvette
that had been preconditioned with this buffer. Apo(a) tryptophan
excitation was performed using a wavelength of 280 nm and a slit width
of 2.5 nm, while tryptophan emission was detected at a wavelength of
340 nm and a slit width of 5 nm with a 290-nm cutoff filter placed in
the emission beam. Ligand solutions, containing 70 nM
r-apo(a) to eliminate dilution effects, were added in a stepwise manner
until saturation of the fluorescence change was attained. The resultant
fluorescence changes were subsequently subjected to non-linear
regression analysis according to equilibrium equations describing one
or two binding sites for
-ACA, as appropriate.
Analytical Ultracentrifugation of 17K
r-Apo(a)--
Sedimentation experiments were performed using a Beckman
XL-I analytical ultracentrifuge with a four-hole rotor and 12-mm carbon-filled epoxy double-sector centerpieces for velocity analysis or
six-channel centerpieces for equilibrium analysis. The temperature was
kept constant at 20 °C. Sedimentation velocity experiments were
performed using a rotor speed of 22,500 rpm. Radial scans, at a
wavelength of 280 nm, were taken at 5-min intervals, and the data were
analyzed with Origin software provided by Beckman-Coulter. Sedimentation equilibrium analyses were preformed at rotor speeds of
4000 and 6000 rpm. The data were analyzed with software provided by
Beckman-Coulter. The buffer utilized was HBS in the presence of 0-100
mM
-ACA. A previously reported value for the partial specific volume of apo(a) (
= 0.69) (21) was used.
Sapp values were converted to
Sw, 20 values by correcting for buffer density
and viscosity using the standard equations (22). The dependence of
Sw, 20 on the concentration of
-ACA was modeled by regression of the data to a simple hyperbolic equation.
Differential Scanning Calorimetry--
All differential scanning
calorimetry experiments were conducted using a VP-DSC calorimeter
(Microcal). Experiments were performed with a scan rate of 90 °C/hr
from 10 °C to 110 °C. 17K r-apo(a) was dialyzed overnight against
4 liters of HBS prior to use. The dialysis buffer was passed through a
0.2-µm filter (Millipore) and used in an initial buffer-buffer scan
to obtain a baseline. At this time, the solutions were replaced and
protein-buffer scans were performed. Apo(a) was used at a concentration
of 0.2 mg/ml in the presence of different concentrations of
-ACA (0, 5, 10, 50, and 100 mM). After correcting for the
buffer-buffer scan and subtracting a progress baseline, the data were
modeled according to a non-two-state model using Origin 5.1 software,
using equations appropriate for either one or two thermal transitions.
The dependence of the ratio of
H*mA and
HmA, corresponding to the
temperature-independent van't Hoff and calorimetric heat changes,
respectively, for the second (
-ACA-dependent) thermal
transition of 17K r-apo(a) on the concentration of
-ACA was modeled
according to a simple hyperbolic relationship.
Purification and Modification of LDL with
5'-Iodoacetamido-fluorescein--
LDL was purified from human plasma
and labeled with 5'-iodoacetamido-fluorescein as previously described
(12). The concentrations of LDL and fluorescently labeled LDL (flu-LDL)
were determined using a modified Bradford assay, and the protein was
stored at 4 °C for no longer than 3 days prior to use.
Binding of 17K r-Apo(a) to flu-LDL and Reversal with
-ACA--
Fluorescein fluorescence measurements of flu-LDL were
performed using an LS50B Luminescence Spectrometer (PerkinElmer Life Sciences). Flu-LDL (50 nM) was titrated with 17K r-apo(a).
Titrations were performed in HBS containing 0.01% Tween 20 in a quartz
cuvette that had been conditioned with this buffer prior to use.
Fluorescein was excited at a wavelength of 495 nm and a slit width of
2.5 nm, while fluorescein emission was detected at a wavelength of 530 nm and a slit width of 5 nm with a 510-nm cutoff filter placed in the
emission beam. Ligand solutions, containing 50 nM flu-LDL to eliminate dilution effects, were added in a stepwise manner until
saturation of the fluorescence change was attained. At this time, the
concentrations of apo(a) (227 nM) and flu-LDL (50 nM) were held constant and
-ACA was titrated until the
reversal of the fluorescence change reached saturation. The
fluorescence changes from the apo(a) titration were subjected to
non-linear regression according to Equation 1,
|
(Eq. 1)
|
where
I is the measured fluorescence change,
dI is the difference between the fluorescence coefficient
for LDL in the free and the apo(a)-bound states, KD
is the dissociation constant for the flu-LDL:apo(a) interaction, and
[A]0 and [B]0 are the total concentrations of 17K r-apo(a) and flu-LDL, respectively. The
reversal of the fluorescence change was modeled independently according
to Equation 2,
|
(Eq. 2)
|
where [E]0 is the total concentration
of
-ACA, KE is the dissociation constant for
the interaction between
-ACA and the site(s) on apo(a) that mediates
non-covalent association to LDL; all of the other parameters are the
same as for Equation 1.
Transient Transfection and Metabolic Labeling of 17K r-Apo(a)
with [35S]Cysteine--
HEK 293 cells, grown to 60%
confluence in 100-mm plates, were transfected with 10 µg of a plasmid
encoding for 17K r-apo(a) using FuGENE 6 transfection reagent (Roche
Molecular Biochemicals) according to the manufacturer's protocol.
After transfection for 6 h, the medium was replaced with 6 ml of
Cys-/Met-DMEM (ICN) and the cells were further incubated for 1 h.
At this time, 300 µCi of [35S]cysteine (PerkinElmer
Life Sciences) was added to each of three plates, and the labeled
protein was allowed to accumulate in the CM for 36 h. After a
brief centrifugation at 1000 × g, the CM was
supplemented with 1 mM phenylmethylsulfonyl fluoride and
concentrated 18-fold using a 100-kDa cutoff centricon (Millipore). The
concentration of 35S-labeled apo(a) in the CM was
determined by enzyme-linked immunosorbent assay using purified apo(a)
as a standard, and the protein was aliquoted and stored at
70 °C
until needed.
Covalent Lp(a) Assembly Assays--
In vitro covalent
Lp(a) assembly assays were performed essentially as previously
described (11). Purified native LDL (50 nm) was incubated with
conditioned media containing 4 nM 35 S-labeled
17K r-apo(a) in the presence of a wide range of
-ACA concentrations
(0, 100 nM, 1 µM, 10 µM, 100 µM, 1 mM, 5 mM, 10 mM, 100 mM, 250 mM, 500 mM) at 37 °C in a total volume of 50 µl. Experiments
in which the concentration of LDL was varied (80, 70, 50, 20, 10 nM), were conducted as described above with a slightly different
-ACA concentration range (0, 1 µM, 10 µM, 100 µM, 1 mM, 5 mM, 10 mM, 25 mM, 100 mM). After a 4-h incubation, the reactions were stopped by
the addition of an equal volume of 2 × Laemmli (23) sample buffer
in the absence of a reducing agent and heated at 95 °C for 5 min.
Samples were then subjected to SDS-PAGE on 4% polyacrylamide gels.
After electrophoresis, the gels were placed in fixing solution (25%
methanol, 12.5% acetic acid in double distilled H2O) for
20 min followed by incubation in Amplify solution (Amersham
Biosciences) for 30 min. The gels were then dried and exposed onto an
Imaging Screen (BioRad) for 16 h. The screen was developed using a
BioRad Molecular Imager FX, and the bands were quantified using
Quantity One 4.0.1 densitometry software. The extent of r-Lp(a)
particle formation was quantified according to the following formula:
% Lp(a) = 100 × [ Lp(a)]/([ Lp(a)] + [ apo(a)]).
The initial rate of covalent Lp(a) assembly (V0)
was estimated by dividing the concentration of Lp(a) by the time of incubation. The initial reaction rates were subsequently modeled with
respect to the concentration of
-ACA using the following relationship in Equation 3,
|
(Eq. 3)
|
where V0 is the initial rate of covalent
Lp(a) formation (nM Lp(a)/hr),
[A]0 and [E]0 are the
total concentrations of 35S-labeled 17K r-apo(a) and
-ACA, and
|
(Eq. 4)
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|
(Eq. 5)
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|
(Eq. 6)
|
|
(Eq. 7)
|
where L is the total concentration of LDL,
KE1 and KE2 are the
dissociation constants for the binding of
-ACA to site 1 and site 2 on apo(a), respectively, KD is the dissociation
constant for the interaction between LDL and apo(a),
KINT is the intrinsic equilibrium constant
between the closed and open conformations of apo(a), and
k1 and k2 represent the
rate constants for covalent Lp(a) particle formation for the closed and
open forms of apo(a), respectively. Equation 3 was derived from first
principles and describes a model (see Fig. 6) in which apo(a) contains
two classes of sites for
-ACA; binding at site(s) 1 inhibits the
non-covalent interaction between apo(a) and apoB-100, while binding at
site(s) 2 is responsible for the enhancement of covalent Lp(a) particle
formation by altering the conformational status of apo(a).
 |
RESULTS AND DISCUSSION |
Binding of
-ACA Results in a Marked Decrease in Apo(a) Intrinsic
Fluorescence--
Titration of a form of recombinant apo(a) consisting
of 17 kringle IV domains followed by the kringle V and protease-like domains (17K r-apo(a)) with
-ACA results in a substantial decrease in intrinsic fluorescence of r-apo(a) as illustrated in Fig.
1. Furthermore, the titration profile
consists of multiple phases (Fig. 1A). To assist in
resolving the phases, we performed a second experiment in which a
construct consisting of only the weak lysine-binding kringles
(KIV5-8) of apo(a) were titrated with
-ACA. Titration of KIV5-8 with
-ACA yielded an increase in tryptophan
fluorescence that followed a simple hyperbolic relationship (Fig.
1B). Non-linear regression of the fluorescence changes
yielded a KD values of 567 ± 49 µM, which is consistent with values previously reported for the weak lysine-binding sites in apo(a) (24, 25). Given the
fluorescence profile observed with KIV5-8, we modeled the
17K r-apo(a) titration as a combination of two classes of
-ACA
binding sites; the first class of sites corresponds to the initial
increase in fluorescence and the second to the subsequent decrease in
fluorescence. Regression of the 17K binding data to the model
corresponding to two classes of
-ACA binding sites resulted in a
very good fit and yielded KD values of 455 ± 20.7 µM and 69 ± 7.3 mM for the first
and second class, respectively (Fig. 1A). It is difficult to
ascertain whether the fluorescence changes observed are attributable to
quenching of tryptophan fluorescence upon binding of
-ACA to
individual kringle domains or whether they are reporting a larger
conformational change that may ensue upon
-ACA binding. To
differentiate between these two alternatives, we used analytical
ultracentrifugation and differential scanning calorimetry to measure
changes in the structural properties of apo(a) in the presence of
-ACA.

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Fig. 1.
Effect of -ACA on
the intrinsic fluorescence of apo(a). A, 17K r-apo(a) (70 nM) was titrated with -ACA, and the resultant changes in
17K intrinsic fluorescence ( ) were measured. The line
represents the result of non-linear regression of the data to an
equation representing binding of -ACA to two independent sites on
17K r-apo(a) described by KD1 and
KD2. B, an identical experiment was
performed using KIV5-8 r-apo(a) except that the
fluorescence changes ( ) were fit to an equation representing binding
of -ACA to a single site on KIV5-8 r-apo(a) described
by KD.
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-ACA Elicits a Marked Conformational Change in
Apo(a)--
Sedimentation velocity experiments demonstrated
that the addition of
-ACA elicits a non-linear decrease in the
sedimentation coefficient of 17K r-apo(a) (Table
I). The change in
Sw, 20 is indicative of a conformational change
in which the hydrodynamic radius (Rh) of apo(a)
increases as the addition of 50 mM
-ACA did not alter
the molecular weight of apo(a) in sedimentation equilibrium experiments
(Table I). Using the change in Sw, 20 (
Sw, 20) as a signal, the data were modeled
according to a simple hyperbolic equation, and a KD
of 8.05 ± 2.77 mM was obtained for the interaction
between
-ACA and the site(s) on apo(a) that is involved in an
intermolecular interaction(s) (Fig. 2).
It is important to note that the dissociation constant obtained using
this approach is an apparent KD
(KD(app)) as the replacement of an internal ligand
by
-ACA is presumably being measured. Interestingly, an apoB-derived
peptide (apoB680-704), which had been previously demonstrated to bind
the weak lysine-binding sites in apo(a) with high affinity (12), had no
effect on the sedimentation coefficient of 17K r-apo(a) (data not
shown). In differential scanning calorimetry (DSC) experiments, the
thermal denaturation of 17K r-apo(a) was characterized by a single
thermal transition (Tm = 55.2 °C), which was mostly reversible
and did not result in an appreciable amount of protein aggregation. The thermal denaturation profile was modeled according to a non-two-state model yielding a temperature-independent calorimetric heat change (
Hm) of 1.2 × 106
kcal/mol, a temperature-independent van't Hoff heat change
(
H*m) of 6.2 × 104
kcal/mol, and a
Hm/
H*m of 19.4. The
ratio
Hm/
H*m is
indicative of the number of structural units undergoing a particular
thermal transition (26). The 17K r-apo(a) derivative contains 19 total domains (18 kringles and 1 protease domain), which is in good agreement
with the experimental value (19.4) obtained. Given the complexity of
17K r-apo(a) (19 domains in total), the single thermal denaturation
transition observed in DSC experiments in the absence of
-ACA
suggests that some of the domains within this protein are involved in
intramolecular interactions. The addition of
-ACA resulted in a
dose-dependent appearance of an additional thermal transition at a melting temperature that saturated at ~69 °C with a
H*mB/
HmB
ratio of ~5.3 (Fig. 3,
A-E), a result that can be accounted for by two alternative
explanations: the binding of
-ACA results in a stabilization in some
of the domains in apo(a) without any accompanying conformational change
and/or the addition of
-ACA leads to a disruption of an
intramolecular interaction, which liberates some of the domains in
apo(a) resulting in the appearance of a second thermal transition. A
simple hyperbolic relationship was used to describe the dependence of
H*mB/
HmB for 17K
r-apo(a) with respect to
-ACA concentration, yielding a
KD(app) value of 7.44 ± 3.06 mM
(Fig. 2). The similarity between the KD(app) values
obtained using the independent approaches of analytical
ultracentrifugation and differential scanning calorimetry (Fig. 2)
suggests that these values represent the same physical phenomenon,
namely the ability of
-ACA to stimulate the conversion of apo(a)
from a closed to an open conformation. We assume that this
conformational change is represented by the substantial decrease in
intrinsic fluorescence that characterizes the titration of 17K r-apo(a)
with
-ACA (Fig. 1A).

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Fig. 2.
Dependence of
Sw, 20 and
HmB/ H*mB
on the concentration of -ACA. The
sedimentation coefficients for 17K r-apo(a) were determined using
analytical ultracentrifugation. Changes in sedimentation coefficients
( Sw, 20) were obtained by subtracting the
Sw, 20 obtained at a given concentration of
-ACA from the value measured in the absence of -ACA.
HmB/ H*mB values
were obtained using a differential scanning calorimeter. The dependence
of Sw, 20 ( ) and
HmB/ H*mB ( ) on
the concentration of -ACA was modeled by regression of the data to
simple hyperbolic equations (solid lines). The affinity of
-ACA for the site(s) on apo(a) that is involved in intramolecular
interactions (KD(app)) was determined from these
analyses.
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Fig. 3.
Differential scanning calorimetry of
apo(a) in the presence of -ACA. The
corrected thermal denaturation profiles were modeled according to a
non-two-state model using Origin 5.1 software. A-E, 17K
r-apo(a) in the presence of 0, 5, 10, 50, and 100 mM
-ACA, respectively. The red lines correspond to the fits
obtained for the data (black lines).
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Effect of
-ACA on the Non-covalent Interaction of Apo(a) and
LDL--
Inhibition of the non-covalent association between apo(a) and
LDL with
-ACA has been demonstrated by numerous reports,
e.g. Ref. 13. In previous studies, however, an immobilized
binding system was utilized. For the purposes of the present study, we wished to assess the efficiency with which
-ACA inhibits
non-covalent Lp(a) assembly in solution so that the data obtained could
be directly compared with subsequent covalent Lp(a) assembly assays that are also performed in solution. To monitor the effect of
-ACA
on the non-covalent association between 17K r-apo(a) and LDL, we
utilized a solution-phase, fluorescence-based system in which LDL was
modified with flu-LDL using sulfhydryl-specific labeling chemistry.
Initially, flu-LDL was titrated with 17K r-apo(a) until the ensuing
fluorescence change reached saturation (Fig. 4). At this time,
-ACA was titrated,
resulting in an incremental recovery of the fluorescence (Fig 4). The
recovery of the fluorescence change was interpreted as a perturbation
of the apo(a)-flu-LDL non-covalent complex attributable to
-ACA
binding to apo(a), as the addition of
-ACA to flu-LDL alone did not
result in an appreciable fluorescence change (data not shown). Note
that covalent association between apo(a) and flu-LDL is not possible
and so bona fide equilibrium binding is observed in this system. The fluorescence changes recorded from both titrations were independently subjected to non-linear regression analyses using Equations 1 and 2. A
dissociation constant (KD) of 23.9 ± 7.2 nM was obtained for the interaction between apo(a) and
flu-LDL, and a dissociation constant (KE) of
326.3 ± 74.3 µM was obtained for the interaction
between
-ACA and the site(s) on 17K r-apo(a) that mediates
non-covalent binding to LDL in the second part of the titration. The
KE obtained using this assay is in good
agreement with the affinity for the interaction between
-ACA and
KIV5-8 measured by intrinsic fluorescence (Fig.
1B) and is consistent with a role for KIV5-8 in
binding to LDL. More importantly, we have clearly demonstrated that the
weak lysine-binding sites in apo(a) KIV5-8 bind
-ACA
with a near identical affinity when expressed in isolation
(KD = 567 ± 49 µM) or in the
context of full-length 17K r-apo(a) (KD = 455 ± 20.7 µM), suggesting that these domains are not
involved intramolecular interactions. Taken together, these data
indicate that the weak lysine-binding sites present within apo(a)
KIV5-8 mediate non-covalent binding to LDL and are
distinct from the site(s) that maintains the closed conformation
of apo(a). Therefore, the binding of LDL does not elicit a
conformational change in the apo(a) molecule, consistent with the
inability of the apoB680-704 peptide to alter the sedimentation
coefficient of apo(a) (see above).

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Fig. 4.
Binding of 17K r-apo(a) to flu-LDL and
reversal with -ACA. Flu-LDL was titrated
with 17K r-apo(a) until the fluorescence change reached saturation, at
which time -ACA was titrated until the reversal of the fluorescence
change reached saturation. The fluorescence changes recorded during the
apo(a) titration ( ) and the reversal of the fluorescence change
measured for the -ACA titration ( ) were independently fit to
Equations 1 and 2, respectively (solid lines), to obtain the
affinity for the non-covalent association between apo(a) and flu-LDL
(KD) and the affinity for -ACA to the site(s) on
apo(a) that binds LDL (KE).
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Low Concentrations of
-ACA Stimulate Covalent Lp(a)
Assembly--
Having demonstrated the efficiency with which
-ACA
inhibits the non-covalent interaction between apo(a) and LDL, we
assessed the effect of
-ACA on the second step of Lp(a) formation by
performing in vitro covalent Lp(a) assembly assays. Fixed
time point (t = 4 h) covalent assembly assays, in
which the
-ACA concentration was systematically varied, were
performed to determine the effective concentration range in which this
compound affected covalent Lp(a) formation. Identical results were
obtained in preliminary experiments using a time course of Lp(a)
formation up to 6 h (data not shown). Interestingly, titration
with
-ACA resulted in a biphasic effect on covalent Lp(a) formation;
low concentrations (100 µM to 1 mM) of
-ACA resulted in an enhancement of the initial rate of covalent Lp(a) particle formation, while higher concentrations (10-500 mM) impeded covalent Lp(a) assembly quite effectively (Fig.
5A). When the effects of
-ACA on non-covalent and covalent Lp(a) assembly are compared (Figs.
4 and 5B), it becomes apparent that the inhibition of
covalent Lp(a) particle formation observed at higher
-ACA concentrations results from the inhibition of non-covalent assembly, reaffirming the notion that the affinity of the non-covalent step of
Lp(a) assembly dictates the efficiency of the covalent step. This
simple two-step model cannot, however, account for the enhancement of
covalent Lp(a) assembly observed with lower
-ACA concentrations that
significantly inhibit the non-covalent step. It is tempting to
speculate that the enhancement of covalent Lp(a) assembly observed with
lower concentrations of
-ACA results from the conversion of apo(a)
from a closed to open conformation. Notably, however, the
concentrations of
-ACA required to elicit a conformational change
(KD(app) ~ 8 mM) in apo(a) do not
precisely coincide with those that promote covalent Lp(a) assembly as
inhibition of Lp(a) assembly is observed beyond ~2 mM
-ACA (Fig. 5B). To reconcile this inconsistency, we
propose that the inhibition of non-covalent Lp(a) assembly due to
binding of KIV5-8 (KD ~ 0.4 mM) is sufficiently great enough at
-ACA
concentrations over 2 mM to subsume the stimulatory effect
through the conformational change. To test this hypothesis, we
systematically increased the LDL:17K ratio (2.5-, 5-, 12.5-, 17.5-, 20-fold) and measured the effect of
-ACA on covalent Lp(a) assembly.
Importantly, increasing the LDL:17K ratio resulted in a saturable
increase in the initial rate of covalent Lp(a) assembly in the absence
of
-ACA and an increase in the magnitude of
-ACA-mediated
covalent Lp(a) assembly enhancement (Fig. 5B). These
observations result from augmenting the proportion of the
KIV5-8 in apo(a) that is bound to apoB-100, thereby
diminishing the ability of
-ACA to inhibit this non-covalent
interaction; as a result, the ability of
-ACA to promote Lp(a)
assembly by eliciting a conformational change in apo(a) is observed
more prominently.

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Fig. 5.
The effect of
-ACA on covalent Lp(a) particle formation.
In vitro covalent Lp(a) assembly assays were carried out in
the presence of increasing concentrations of -ACA. A,
Lp(a) was separated from free apo(a) using SDS-PAGE and visualized by
autoradiography. The percent Lp(a) formed was determined using
densitometry to measure the amount of Lp(a) and free apo(a) at a given
concentration of -ACA. B, covalent Lp(a) assembly assays
were performed in which the concentration of LDL was varied (10 nM, ; 20 nM, ; 50 nM, ; 70 nM, ; 80 nM, ). The initial velocity of
covalent Lp(a) particle formation (V0), which
was calculated from the measured percent Lp(a), was modeled with
respect to the concentration of -ACA using Equation 3. The lines
represent the fit obtained for each LDL concentration when the data
were subjected to a global fit (red line, 10 nM;
green line, 20 nM; blue line, 50 nM; pink line, 70 nM; cyan
line, 80 nM) using this equation, which describes the
novel model for Lp(a) assembly shown in Fig. 6. Inset, to
facilitate comparison of the effect of -ACA on covalent Lp(a)
assembly and non-covalent binding of apo(a) and LDL, the calculated
effect of -ACA on the latter is also shown for each of the LDL
concentrations used (red line, , 10 nM;
green line, , 20 nM; blue line,
, 50 nM; pink line, , 70 nM;
cyan line, , 80 nM). The effect of -ACA on
non-covalent binding was calculated using KD and
KE, measured in the fluorescence-based
non-covalent binding assay, along with the total concentrations of
apo(a), LDL, and -ACA used in the covalent Lp(a) assembly
assays.
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|
The dependence of the initial rate of covalent Lp(a) particle formation
on both the concentration of
-ACA and the LDL:17K ratio, combined
with our demonstration of open and closed conformations of apo(a) led
to the development of a new model for Lp(a) assembly that takes into
account the conformational status of apo(a) (schematized in Fig.
6 and described by Equation 3). The
solid lines in Fig. 5B are regression lines based
on a global fit of the covalent assembly data to Equation 3. The
dissociation constants and rate constants that characterize each step
of the model (k1 = 0.0497 ± 0.0106 h
1, k2 = 0.3040 ± 0.118 h
1, KINT = 0.025 ± 0.015, KD = 21.1 ± 5.5 nM,
KE1 = 543 ± 126.8 µM, and
KE2 = 57.1 ± 10.1 µM) are
displayed in Fig. 6. In the absence of
-ACA, the majority of 17K
r-apo(a) is in the closed conformation (KINT = [open]/[closed] = 0.025 ± 0.015). The closed form of apo(a)
is capable of interacting with LDL (KD = 21.1 ± 5.5 nM) to form a non-covalent Lp(a) complex, which
subsequently becomes converted to covalent Lp(a) with a rate constant
(k1) of 0.0497 ± 0.0106 h
1.
The addition of
-ACA elicits two opposing effects on Lp(a) assembly
that are mediated by two distinct sites on apo(a); binding at site 1 (KE1 = 543 ± 126.8 µM)
inhibits covalent Lp(a) assembly by competing with LDL for binding to
KIV5-8 in apo(a). In contrast, binding at site 2 (KE2 = 57.1 ± 10.1 µM)
increases the rate of covalent Lp(a) assembly
(k2 = 0.304 ± 0.118 h
1) by
competing with the internal ligand for binding to site 2 and thereby
promoting the open conformation of apo(a). The relatively high affinity
binding that characterizes the interaction between
-ACA and site 2 on apo(a) (KE2 = 57.1 ± 10.1 µM) is suggestive of a role for apo(a) KIV10
as we have previously demonstrated that KIV10 binds
-ACA
with a comparable affinity (KD = 33 ± 4 µM) (24). Furthermore, preliminary experiments using a
17K r-apo(a) derivative in which the lysine-binding pocket in KIV10 has been mutated abrogates the
-ACA-mediated
enhancement of covalent Lp(a)
assembly.2

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Fig. 6.
A novel model for Lp(a) assembly.
Based on the data accumulated in this study, we amended the simple
two-step model for Lp(a) assembly. In this new model, apo(a) contains
two distinct lysine-binding sites: one that mediates non-covalent
binding to LDL (site 1) and the other that is involved in an
intramolecular interaction with an internal ligand thereby maintaining
the closed conformation of apo(a) (site 2). The dissociation constants
and rate constants were obtained from a global fit of the data to
Equation 3. In the present study, we have provided evidence that
-ACA binding sites 1 and 2 on apo(a) are represented by the weak
lysine-binding sites present within KIV5-8 and the strong
lysine-binding site within KIV10 respectively. We have
depicted the intramolecular interaction between the strong
lysine-binding site in KIV10 and, for convenience, the C
terminus of apo(a).
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This model is strongly supported by the good agreement between the
dissociation constants obtained through physical measurements and those
obtained from modeling the covalent Lp(a) assembly data to Equation 3.
The affinity obtained for the non-covalent interaction between apo(a)
and LDL (KD = 21.1 ± 5.5 nM) is in
good agreement with the value that was measured using extrinsic
fluorescence (KD = 23.9 ± 7.2 nM;
Fig. 4). The affinity obtained for the interaction between
-ACA and
site 1 on 17K r-apo(a) (KE1 = 543 ± 126.8 µM) agrees with that measured for 17K and
KIV5-8 using intrinsic fluorescence
(KD = 455 ± 20.7 µM and 567 ± 49 µM, respectively; Fig. 1, A and
B) and that obtained in the extrinsic fluorescence assay
(KE = 326.3 ± 74.3 µM; Fig. 4). Furthermore, these findings indicate that site 1 on apo(a) corresponds to KIV5-8. The apparent affinity of
-ACA
for site 2 on 17K r-apo(a) (KE2(app) = KE2/KINT = 2.3 ± 1.4 mM) obtained from the modeling is consistent with the
apparent affinity measured by analytical ultracentrifugation and DSC
(KD(app) = 8.05 ± 2.77 mM and
7.44 ± 3.06 mM, respectively; Fig. 2). Finally, the
rate constant describing covalent Lp(a) formation from the open form of
apo(a) (k2; Fig. 6) exceeds that for the closed
conformation by a factor of 6. As such, the data clearly demonstrate
that the
-ACA-induced conformational change in apo(a) is
responsible for the enhanced rate of covalent Lp(a) assembly. The
mechanism by which the conformational change in apo(a) enhances Lp(a)
assembly remains to be elucidated, but may involve either (i) promoting
access to the free cysteine in apo(a) KIV9 by alleviation
of steric hindrance and/or (ii) facilitating a more favorable
presentation of the free cysteine in apo(a) KIV9. In
addition, our model predicts that the enhancing effect of
-ACA is
independent of an effect on non-covalent binding of apo(a) to LDL.
The physical characteristics and functional implications of the
conformational change for apo(a) presented in this report are
remarkably analogous to those previously described for plasminogen. Plasminogen is kept in a closed conformation via an intramolecular interaction between the N-terminal tail domain and kringle V (27).
-ACA replaces the internal ligand and leads to a transition from a
closed to open conformation, resulting in a change in tryptophan fluorescence (28), an increase in the radius of gyration (18), and an
enhancement of tPA- or uPA-mediated plasminogen activation to plasmin
(29). Binding of plasminogen to fibrin, an important cofactor for
plasminogen activation, has been postulated to elicit this
conformational change (30). Surprisingly, the domains that are
responsible for maintaining apo(a) and plasminogen in closed conformations have not been conserved as apo(a) does not contain an
N-terminal tail domain. Furthermore, our preliminary data suggest a
role for the strong lysine-binding site in apo(a)
KIV10 in maintaining apo(a) in a closed conformation. Taken
together, this suggests that this mode of conformational regulation may
be a paradigm that is applicable to other kringle-containing proteins.
In this regard, closed conformations, stabilized by intramolecular
domain-domain interactions, have also been reported in other kringle-
(or kringle-like)-containing proteins such as prothrombin (31, 32),
hepatocyte growth factor/scatter factor (33, 34), and fibronectin (35,
36). The closed conformation in these functionally diverse proteins
provides a mechanism for functional regulation by masking important
binding sites that are required for the functions of the respective
proteins. In the case of apo(a), the conformational switch from the
closed to open form enhances its covalent binding to the apoB-100
component of LDL to form Lp(a) particles. In the case of plasminogen,
the conformational change accelerates the rate of tPA- or uPA-mediated plasminogen activation to plasmin. For fibronectin, the conformational change may regulate extracellular matrix function by affecting cell
adhesion. Although the relevance of the closed conformations in
prothrombin and hepatocyte growth factor/scatter factor are poorly
understood, it is reasonable to hypothesize that the transition to the
open conformation may have functional implications.
It is tempting to speculate that the involvement of a kringle (or
kringle-like) domain(s) in intramolecular interactions may keep
proteins in non-reactive forms that require conformational changes for
their respective activities. Our findings raise the novel possibility
that the efficiency of Lp(a) assembly in vivo can be
modulated by an accessory protein(s) that alters the conformational status of apo(a) and that this accessory protein(s) may be localized on
the hepatocyte surface where Lp(a) assembly has been postulated to
occur. We would expect that the conformational change in apo(a) would
be elicited much more efficiently by a lysine residue(s) in an
accessory protein(s) than by free lysine or
-ACA because the
presence of additional sequences would likely increase both the
affinity and specificity of the binding interaction. In support of this
notion, the weak lysine-binding sites in apo(a) display a 10,000-fold
higher affinity for a lysine residue(s) in the context of the apoB
molecule compared with either free lysine or
-ACA (12).
 |
FOOTNOTES |
*
This work was supported by Canadian Institutes for Health
Research Grant 11271 (to M. L. K.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Career Investigator of the Heart and Stroke Foundation of Ontario.
To whom correspondence should be addressed. Tel.: 613-533-6586; Fax:
613-533-2987; E-mail: mk11@post.queensu.ca.
Published, JBC Papers in Press, February 3, 2003, DOI 10.1074/jbc.M212855200
2
M. L. Koschinsky and L. Becker, unpublished data.
 |
ABBREVIATIONS |
The abbreviations used are:
Lp(a), lipoprotein(a);
LDL, low density lipoprotein;
apo(a), apolipoprotein(a);
apoB, apolipoproteinB;
-ACA,
-aminocaproic
acid;
KIV, kringle IV;
K, kringle;
r-apo(a), recombinant apo(a);
HEK, human embryonic kidney;
CM, conditioned media;
HBS, HEPES-buffered
saline;
flu-LDL, fluorescently labeled LDL;
DSC, differential scanning
calorimetry.
 |
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