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. KoschinskyDagger

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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 epsilon -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 epsilon -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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 epsilon -aminocaproic acid (epsilon -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 epsilon -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 epsilon -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 epsilon -ACA on the structure of apo(a). Fluorescence spectroscopy, analytical ultracentrifugation, and differential scanning calorimetry were used to determine whether apo(a) undergoes a epsilon -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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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 epsilon -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 epsilon -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 epsilon -ACA. A previously reported value for the partial specific volume of apo(a) (rho  = 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 Delta Sw, 20 on the concentration of epsilon -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 epsilon -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 Delta H*mA and Delta HmA, corresponding to the temperature-independent van't Hoff and calorimetric heat changes, respectively, for the second (epsilon -ACA-dependent) thermal transition of 17K r-apo(a) on the concentration of epsilon -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 epsilon -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 epsilon -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,


&Dgr;I=0.5*dI*(K<SUB>D</SUB>+[A]<SUB>0</SUB>+[B]<SUB>0</SUB> (Eq. 1)

−<RAD><RCD>(K<SUB>D</SUB>+[A]<SUB>0</SUB>+[B]<SUB>0</SUB>)<SUP>2</SUP>−4[A]<SUB>0</SUB>[B]<SUB>0</SUB></RCD></RAD>)
where Delta 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,
&Dgr;I=0.5*dI*<FENCE>K<SUB>D</SUB>+[A]<SUB>0</SUB>+[B]<SUB>0</SUB>+<FR><NU>[E]<SUB>0</SUB>K<SUB>D</SUB></NU><DE>K<SUB>E</SUB></DE></FR></FENCE> (Eq. 2)

<FENCE>−<RAD><RCD><FENCE>K<SUB>D</SUB>+[A]<SUB>0</SUB>+[B]<SUB>0</SUB>+<FR><NU>[E]<SUB>0</SUB>K<SUB>D</SUB></NU><DE>K<SUB>E</SUB></DE></FR></FENCE><SUP>2</SUP>−4[A]<SUB>0</SUB>[B]<SUB>0</SUB></RCD></RAD></FENCE>
where [E]0 is the total concentration of epsilon -ACA, KE is the dissociation constant for the interaction between epsilon -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 epsilon -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 epsilon -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 epsilon  -ACA using the following relationship in Equation 3,


<FR><NU>V<SUB>0</SUB></NU><DE>[A]<SUB>0</SUB></DE></FR>=<FR><NU>A+B[E]<SUB>0</SUB></NU><DE>C+D[E]<SUB>0</SUB>+[E]<SUB>0</SUB><SUP>2</SUP></DE></FR> (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 epsilon -ACA, and
A=<FR><NU>LK<SUB>E1</SUB>K<SUB>E2</SUB></NU><DE>K<SUB>D</SUB></DE></FR><FENCE><FR><NU>k<SUB>1</SUB></NU><DE>K<SUB>INT</SUB></DE></FR>+k<SUB>2</SUB></FENCE> (Eq. 4)

B=<FR><NU>Lk<SUB>2</SUB>K<SUB>E1</SUB></NU><DE>K<SUB>D</SUB></DE></FR> (Eq. 5)

C=K<SUB>E1</SUB>K<SUB>E2</SUB><FENCE>1+<FR><NU>1</NU><DE>K<SUB>INT</SUB></DE></FR>+<FR><NU>L</NU><DE>K<SUB>D</SUB></DE></FR>+<FR><NU>L</NU><DE>K<SUB>D</SUB>K<SUB>INT</SUB></DE></FR></FENCE> (Eq. 6)

D=K<SUB>E2</SUB>+K<SUB>E1</SUB>+<FR><NU>K<SUB>E2</SUB></NU><DE>K<SUB>INT</SUB></DE></FR>+<FR><NU>K<SUB>E1</SUB>L</NU><DE>K<SUB>D</SUB></DE></FR> (Eq. 7)
where L is the total concentration of LDL, KE1 and KE2 are the dissociation constants for the binding of epsilon -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 epsilon -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
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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Binding of epsilon -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 epsilon -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 epsilon -ACA. Titration of KIV5-8 with epsilon -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 epsilon -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 epsilon -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 epsilon -ACA to individual kringle domains or whether they are reporting a larger conformational change that may ensue upon epsilon -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 epsilon -ACA.


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Fig. 1.   Effect of epsilon -ACA on the intrinsic fluorescence of apo(a). A, 17K r-apo(a) (70 nM) was titrated with epsilon -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 epsilon -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 epsilon -ACA to a single site on KIV5-8 r-apo(a) described by KD.

epsilon -ACA Elicits a Marked Conformational Change in Apo(a)-- Sedimentation velocity experiments demonstrated that the addition of epsilon -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 epsilon -ACA did not alter the molecular weight of apo(a) in sedimentation equilibrium experiments (Table I). Using the change in Sw, 20 (Delta 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 epsilon -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 epsilon -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 (Delta Hm) of 1.2 × 106 kcal/mol, a temperature-independent van't Hoff heat change (Delta H*m) of 6.2 × 104 kcal/mol, and a Delta Hm/Delta H*m of 19.4. The ratio Delta Hm/Delta 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 epsilon -ACA suggests that some of the domains within this protein are involved in intramolecular interactions. The addition of epsilon -ACA resulted in a dose-dependent appearance of an additional thermal transition at a melting temperature that saturated at ~69 °C with a Delta H*mB/Delta HmB ratio of ~5.3 (Fig. 3, A-E), a result that can be accounted for by two alternative explanations: the binding of epsilon -ACA results in a stabilization in some of the domains in apo(a) without any accompanying conformational change and/or the addition of epsilon -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 Delta H*mB/Delta HmB for 17K r-apo(a) with respect to epsilon -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 epsilon -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 epsilon -ACA (Fig. 1A).


                              
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Table I
Analytical ultracentrifugation of 17K r-apo(a) in the presence of varepsilon -ACA


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Fig. 2.   Dependence of Sw, 20 and Delta HmB/Delta H*mB on the concentration of epsilon -ACA. The sedimentation coefficients for 17K r-apo(a) were determined using analytical ultracentrifugation. Changes in sedimentation coefficients (Delta Sw, 20) were obtained by subtracting the Sw, 20 obtained at a given concentration of epsilon -ACA from the value measured in the absence of epsilon -ACA. Delta HmB/Delta H*mB values were obtained using a differential scanning calorimeter. The dependence of Delta Sw, 20 (open circle ) and Delta HmB/Delta H*mB () on the concentration of epsilon -ACA was modeled by regression of the data to simple hyperbolic equations (solid lines). The affinity of epsilon -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 epsilon -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 epsilon -ACA, respectively. The red lines correspond to the fits obtained for the data (black lines).

Effect of epsilon -ACA on the Non-covalent Interaction of Apo(a) and LDL-- Inhibition of the non-covalent association between apo(a) and LDL with epsilon -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 epsilon -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 epsilon -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, epsilon -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 epsilon -ACA binding to apo(a), as the addition of epsilon -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 epsilon -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 epsilon -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 epsilon -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 epsilon -ACA. Flu-LDL was titrated with 17K r-apo(a) until the fluorescence change reached saturation, at which time epsilon -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 epsilon -ACA titration (open circle ) 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 epsilon -ACA to the site(s) on apo(a) that binds LDL (KE).

Low Concentrations of epsilon -ACA Stimulate Covalent Lp(a) Assembly-- Having demonstrated the efficiency with which epsilon -ACA inhibits the non-covalent interaction between apo(a) and LDL, we assessed the effect of epsilon -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 epsilon -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 epsilon -ACA resulted in a biphasic effect on covalent Lp(a) formation; low concentrations (100 µM to 1 mM) of epsilon -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 epsilon -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 epsilon -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 epsilon -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 epsilon -ACA results from the conversion of apo(a) from a closed to open conformation. Notably, however, the concentrations of epsilon -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 epsilon -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 epsilon -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 epsilon -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 epsilon -ACA and an increase in the magnitude of epsilon -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 epsilon -ACA to inhibit this non-covalent interaction; as a result, the ability of epsilon -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 epsilon -ACA on covalent Lp(a) particle formation. In vitro covalent Lp(a) assembly assays were carried out in the presence of increasing concentrations of epsilon -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 epsilon -ACA. B, covalent Lp(a) assembly assays were performed in which the concentration of LDL was varied (10 nM, ; 20 nM, black-triangle; 50 nM, black-square; 70 nM, black-down-triangle ; 80 nM, black-diamond ). 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 epsilon -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 epsilon -ACA on covalent Lp(a) assembly and non-covalent binding of apo(a) and LDL, the calculated effect of epsilon -ACA on the latter is also shown for each of the LDL concentrations used (red line, , 10 nM; green line, black-triangle, 20 nM; blue line, black-square, 50 nM; pink line, black-down-triangle , 70 nM; cyan line, black-diamond , 80 nM). The effect of epsilon -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 epsilon -ACA used in the covalent Lp(a) assembly assays.

The dependence of the initial rate of covalent Lp(a) particle formation on both the concentration of epsilon -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 epsilon -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 epsilon -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 epsilon -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 epsilon -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 epsilon -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 epsilon -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).

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 epsilon -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 epsilon -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 epsilon -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 epsilon -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). epsilon -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 epsilon -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 epsilon -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.

Dagger 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; epsilon -ACA, epsilon -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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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