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 binding to low-density lipoprotein receptors on HepG2 cells Seyed A Mousavi1, Knut E Berge1, Trond Berg2 and Trond P Leren1 Unit for Cardiac and Cardiovascular Genetics, Department of Medical Genetics, Oslo University Hospital Rikshospitalet, Norway Department of Molecular Biosciences, University of Oslo, Norway Keywords association; dissociation; dissociation constants; low-density lipoprotein receptor; proprotein convertase subtilisin ⁄ kexin 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 June 2011, accepted 16 June 2011) doi:10.1111/j.1742-4658.2011.08219.x Proprotein convertase subtilisin ⁄ kexin type (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 ([125I]TC-PCSK9), we measured the affinity and kinetics of binding of PCSK9 to LDLR on HepG2 cells at °C The extent of [125I]TC-PCSK9 binding increased as cell surface LDLR density increased Unlabeled wild-type and two gain-of-function mutants of PCSK9 reduced binding of [125I]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 fitted the data The time course of [125I]TC-PCSK9 binding showed two phases in the association kinetics Dissociation of [125I]TC-PCSK9 also occurred in two phases Unlabeled PCSK9 accelerated the dissociation of [125I]TC-PCSK9 At low pH, only one phase of dissociation was apparent Furthermore, the dissociation of [125I]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 (PCSK9) is a protein secreted by the liver that was recognized as an important regulator of cholesterol homeostasis through its link to autosomal dominant hypercholesterolemia [1–3] Central to its role as a cholesterol-regulatory 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 cholesterol 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 S A Mousavi et al Characterization of the binding of PCSK9 to intact cells levels of plasma LDL cholesterol, whereas loss-of-function 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 overexpression of PCSK9 in the liver results in increased plasma LDL cholesterol levels, whereas knockout of the PCSK9 gene decreases plasma levels of LDL cholesterol [4–6,13] (for a recent review see [14]) PCSK9 exerts its action by interacting with a binding 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 residues 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 ([125I]TC-PCSK9) binding to HepG2 cells Incubation of cells in lipoprotein-free medium increased 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 medium (Fig 1A) Under both conditions, the amount of [125I]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 [125I]TC-PCSK9 (Fig 1B) The binding of [125I]TC-PCSK9-D374Y is presented for comparison Approximately five times less [125I]TC-PCSK9-D374Y than [125I]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 associated with cells was at least 10 times of the counts associated with blank wells Fig Cell density dependence of the binding of [125I]TC-PCSK9 and [125I]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 125I-labeled anti-(rabbit IgG) to cells, and the specific binding of [125I]TC-PCSK9 (5 lgỈmL)1, 70 nM) (B) and [125I]TCPCSK9-D374Y (1 lgỈmL)1, 14 nM) (C) to cells were determined as described in Experimental procedures Mean cell numbers in wells seeded with the highest cell density were 7.55 · 105 and 7.5 · 105 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 single experiment Similar results were obtained in several independent 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 FEBS Journal 278 (2011) 2938–2950 ª 2011 The Authors Journal compilation ª 2011 FEBS 2939 Characterization of the binding of PCSK9 to intact cells S A Mousavi et al As the gain-of-function D374Y mutation is localized in the LDLR-binding region of PCSK9, the enhanced binding of [125I]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 [125I]TC-PCSK9 and [125I]TC-PCSK9-D374Y to HepG2 cells To further demonstrate the specificity of [125I]TCPCSK9 binding, we incubated the cells for 30 at °C in the presence of different unlabeled ligands prior to incubating the cells with [125I]TC-PCSK9 The inclusion of a 200-fold excess of unlabeled PCSK9D374Y reduced binding by 82–90% (depending on the batch used) Unlabeled wild-type PCSK9 and PCSK9S127R were also able to reduce the binding of [125I]TC-PCSK9 (see below) TLDLR possesses distinct binding domains for apoB-100 (the main apolipoprotein in LDL) and PCSK9 In agreement with previous studies showing that LDL can inhibit the uptake of PCSK9 in cells [19,20], binding of [125I]TC-PCSK9 was inhibited by > 60% when unlabeled LDL (1 mgỈmL)1) was included in the incubation medium (data not shown), presumably because of steric blocking of the adjacent EGF-A domain Incubation with formaldehyde-treated BSA at a concentration sufficient to saturate scavenger receptors (1 mgỈmL)1) [21] had little effect on specific [125I]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 [125I]TC-PCSK9 binding to HepG2 cells Moreover, large amounts of unlabeled PCSK9 (wild type and D374Y) were required to determine nonspecific binding These technical limitations precluded determination of equilibrium dissociation constants (Kd) for [125I]TC-PCSK9 and [125I]TC-PCSK9-D374Y In order to estimate the binding affinities of PCSK9 and PCSK9-D374Y for HepG2 cell LDLR, we incubated the cells with a fixed concentration of [125I] TC-labeled ligand in the presence of increasing concentrations of the unlabeled counterpart (Fig 2B,C) The abilities of unlabeled PCSK9-D374Y and PCSK9S127R to reduce [125I]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 Scatchard Nonlinear regression analysis of data from individual binding curves indicated that the data were 2940 described best by a two-binding site model The IC50 (the concentration of the competing ligand that inhibits 50% of the specific binding of [125I]TC-PCSK9) values 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 ([125I]TC-PCSK9D374Y) 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 Kd values obtained for the higher-affinity and lower-affinity binding sites are listed in Table 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 cooperativity 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 coefficient < 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 significantly different from unity (0.87 ± 0.07, n = 3) (not shown) Kinetic characteristics of [125I]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 [125I]TC-PCSK9 and [125I]TC-PCSK9-D374Y dissociation from and association with HepG2 cells were determined Kinetic association The time course of [125I]TC-PCSK9 association with HepG2 cells at °C is shown in Fig 3A,B Specific binding of [125I]TC-PCSK9 (5 lgỈmL)1, 70 nm) to cells FEBS Journal 278 (2011) 2938–2950 ª 2011 The Authors Journal compilation ª 2011 FEBS S A Mousavi et al A B C reached an apparent equilibrium within h Binding of [125I]TC-PCSK9-D374Y (1 lgỈmL)1, 14 nm) to HepG2 cells indicates that [125I]TC-PCSK9-D374Y at a concentration five times lower than that of [125I]TCPCSK9 bound to the cells in a similar time-dependent manner, and approached binding equilibrium at nearly the same rate, suggesting a higher affinity of [125I]TCPCSK9-D374Y for cell surface LDLR For both [125I]TC-PCSK9 and [125I]TC-PCSK9-D374Y, the time courses of binding were biphasic, and data from individual association curves were well fitted by a twophase exponential association model, suggesting that surface binding has two components, one rapid and one slow The half-time for association of [125I]TCPCSK9 with the rapid component, representing 35% (± 5.3%) of specific equilibrium binding, was 6.6 (± 1.03 min), whereas the half-time for binding to the Characterization of the binding of PCSK9 to intact cells Fig Nonlinear regression and Scatchard analyses of binding-inhibition data (A) Inhibition of [125I]TC-PCSK9 (2.5 lgỈmL)1) binding to HepG2 cells ( 9.8 · 105) 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 determinations 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 [125I]TC-PCSK9 binding to HepG2 cells (9.2–9.8 · 105) by increasing concentrations of unlabeled wild-type PCSK9 Error bars are standard 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 [125I]TC-PCSK9 binding by unlabeled wild-type PCSK9 (C) Inhibition of [125I]TCPCSK9-D374Y (1 lgỈmL)1) binding to HepG2 cells (9.2–9.8 · 105) 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 [125I]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 [125I]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 (IC50, Kd) determined from these experiments (means ± standard deviations) are shown in Table The asterisk indicates the range of duplicate determinations from a single experiment slow component was 94 (± 23 min) (n = 3) The corresponding half-times for [125I]TC-PCSK9-D374Y binding were 6.1 (± 0.8 min) and 89 (± min) (n = 3), respectively The observed association rate constants kobs for the rapid phase [kobs(rapid)] and for the slow phase [kobs(slow)] are shown in Table Kinetic dissociation The data in Fig show the time course of [125I]TCPCSK9 and [125I]TC-PCSK9-D374Y dissociation from HepG2 cells It is evident that the dissociation of both [125I]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 [125I]TC-PCSK9 dissociated during the rapid phase, with a half-time of 19 (± 2.7 min), and the remaining bound [125I]TC-PCSK9 dissociated slowly, with a half-time of 270 (± 22 min) (n = 3) The FEBS Journal 278 (2011) 2938–2950 ª 2011 The Authors Journal compilation ª 2011 FEBS 2941 Characterization of the binding of PCSK9 to intact cells S A Mousavi et al Table Parameters obtained from binding-inhibition experiments Best-fit values for IC50 were derived from nonlinear regression analysis Kd 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 values ± standard deviations were calculated from these values IC50 (nM) Kd (nM) Labeled PCSK9 Competitive ligand High Low High Low [125I]TC-PCSK9 [125I]TC-PCSK9 [125I]TC-PCSK9 [125I]TC-PCSK9-D374Y PCSK9-wild type (3) PCSK9-S127R (1) PCSK9-D374Y (1) PCSK9-D374Y (3) 374 ± 82 263 68.5 92 ± 17 2500 ± 400 1900 1540 1200 ± 300 626 ± 113 548 107 125 ± 20 2800 ± 300 2900 1250 1600 ± 200 slowly, with a half-time of 297 (± 25 min) (n = 3) The fraction of [125I]TC-PCSK9-D374Y that remained bound after h was about 45% The dissociation rate constants [koff] for the rapid phase [koff(rapid)] and for the slow phase [koff(slow)] are shown in Table Taken together with the association data, these results suggest that the increased affinity of PCSK9D374Y for cell surface LDLR is mainly determined by the rate of association It should be noted that quantitative 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 estimation of the association rate constant [kon] and the kinetic Kd (i.e the Kd representing the koff ⁄ kon ratio) Dissociation of [125I]TC-PCSK9 in the presence of unlabeled PCSK9 Fig Association time courses of the binding of [125I]TC-PCSK9 and [125I]TC-PCSK9-D374Y to HepG2 cells at °C Cells ( 9.2– 9.5 · 105) were incubated for the indicated times in binding medium containing [125I]TC-PCSK9 (5 lgỈmL)1) or [125I]TC-PCSK9D374Y (1 lgỈmL)1) At each time point, the cells were washed, and the specific binding was determined (A) Binding presented as percentage of total radioactivity added (B) Binding presented as the amount of [125I]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 106 cells) The curves were fitted with the two-phase exponential association model amount of [125I]TC-PCSK9 that remained bound after h was about 40% Approximately 20% of the bound [125I]TC-PCSK9-D374Y dissociated rapidly, with a half-time of 21 (± 3.7 min), and the remaining bound [125I]TC-PCSK9-D374Y dissociated more 2942 In order to establish whether the [125I]TC-PCSK9 bound to the cells after h was dissociable, dissociation experiments 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 halftime of the rapid phase was reduced from 19 to 16 and the half-time of the slow phase was reduced from 270 to 154 In the presence of unlabeled PCSK9D374Y, the half-time of the rapid phase of [125I]TCPCSK9-D374Y dissociation was reduced from 21 to 19 min, and the half-time of the slow phase was reduced from 297 to 179 (Fig 4B) These results suggest that the reason for ligand remaining bound to cells after h is not irreversibility of the binding Dissociation of [125I]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 FEBS Journal 278 (2011) 2938–2950 ª 2011 The Authors Journal compilation ª 2011 FEBS S A Mousavi et al Characterization of the binding of PCSK9 to intact cells Table Parameters obtained from kinetic experiments koff values were obtained by fitting the time course data with a two-exponential decay phase model koff(rapid) and koff(slow) are the dissociation rate constants for the rapid and slow dissociation components, respectively kobs values were obtained by fitting the time course data with a two-exponential association model kobs(rapid) and kobs(slow) are the observed association rate constants for the rapid and slow association phases, respectively The values for the constants are means ± standard deviations of three experiments The data from each experiment were analyzed separately The concentrations used are those described in the text koff (min)1) Labeled ligand 125 [ I]TC-PCSK9 [125I]TC-PCSK9-D374Y kobs (min)1) koff(rapid) koff(slow) kobs(rapid) kobs(slow) 0.036 ± 0.005 0.033 ± 0.006 0.0026 ± 0.0002 0.0023 ± 0.0002 0.108 ± 0.016 0.116 ± 0.017 0.0077 ± 0.0020 0.0079 ± 0.0008 [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 complex at endosomal pH [15] However, the rates of dissociation 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 [125I]TC-PCSK9 was measured at pH 6.2 (to mimic the early endosomal pH) As shown in Fig 4A, low pH did indeed markedly reduce the dissociation of [125I]TC-PCSK9 from cells Dissociation occurred as a monophasic process with a rate constant [koff] of 0.0009 ± 0.00012 min)1 (n = 3), which corresponds to a half-time of dissociation of 770 Lowering the pH of dissociation medium to 6.2 also led to monophasic and slow dissociation of [125I]TC-PCSK9-D374Y with a halftime of 810 (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 dissociation of ligand from receptor The reduced dissociation 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 [125I]TC-PCSK9 The dependence of dissociation of [125I]TC-PCSK9 and [125I]TC-PCSK9-D374Y on the length of association 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 ( : for [125I]TCPCSK9 and  : for [125I]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 [125I]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, respectively The dependence of the kinetics of [125I]TCPCSK9-D374Y dissociation on the length of binding time was also examined Again, dissociation from the rapid component was found to be faster at 60 than at 240 (36% versus 19% under equilibrium conditions) (Fig 5B) These data suggest that there is a fraction of rapidly dissociating receptors,  40% after 60 of association, that converts with time to a receptor state that releases bound ligand very slowly The size of the fraction 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 process, 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 binding 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 lipoproteins, and there was a corresponding increase in the binding of [125I]TC-PCSK9 and [125I]TC-PCSK9D374Y Second, the extent of specific binding of both [125I]TC-labeled ligands was a linear function of the cell density Finally, the binding is specific, as the FEBS Journal 278 (2011) 2938–2950 ª 2011 The Authors Journal compilation ª 2011 FEBS 2943 Characterization of the binding of PCSK9 to intact cells S A Mousavi et al Fig Dissociation time courses and effects of unlabeled ligands and low pH on the dissociation rates of [125I]TC-PCSK9 (A) and [125I]TC-PCSK9-D374Y (B) from HepG2 cells Binding to equilibrium and removal of unbound [125I]TC-labeled ligands were performed as described in Experimental procedures The cells were then incubated 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 presented 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 dissociation at low pH was best described by a one-exponential decay phase model Final concentrations of unlabeled ligands in the dissociation medium were 120 lgỈmL)1 (wild-type PCSK9) and 30 lgỈmL)1 (PCSK9-D374Y) If the Kd 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 concentrations of unlabeled ligands used binding of [125I]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 [125I]TC-PCSK9 for the same receptor Linear [27–29] and curvilinear [30,31] Scatchard plots for LDL binding to LDLR are observed Analysis of inhibition curves of [125I]TC-PCSK9 and [125I]TC-PCSK9-D374Y by the Scatchard method 2944 Fig Dissociation kinetics of [125I]TC-PCSK9 (A) and [125I]TCPCSK9-D374Y (B) as a function of time of association: HepG2 cells ( · 105) were incubated at °C in medium containing [125I]TClabeled ligand for 60 or 240 After removal of unbound [125I]TClabeled ligand, the cells were incubated at °C in fresh medium, and dissociation was measured as described in Experimental procedures 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 consistently showed curvilinear plots that implicated the presence of high-affinity and low-affinity sites ⁄ states with affinities for PCSK9 that differ approximately 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 simple bimolecular reaction (see Doc S1, model A) One possibility that could explain the observed curvilinear 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 [125I]TClabeled ligands were employed Moreover, we believe that the [125I]TC-labeling method, in contrast to the direct 125I-labeling method, does not appreciably alter the binding properties of PCSK9, as [125I]TC-PCSK9- FEBS Journal 278 (2011) 2938–2950 ª 2011 The Authors Journal compilation ª 2011 FEBS S A Mousavi et al D374Y consistently displayed a much higher affinity for HepG2 cell surface receptors than did [125I]TCPCSK9 Binding to sites other than LDLR, such as LDLR related protein (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 cholesterol levels [33], and therefore cannot account for the observed increased binding of [125I]TC-PCSK9 to HepG2 cells grown in the absence of lipoproteins It is more likely that the observed heterogeneity in binding of [125I]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 Kd values reported for wildtype 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 Kd of wild-type PCSK9 for the high-affinity LDLRs (626 ± 113 nm) in intact cells estimated from Scatchard plots is within this range The calculated Kd value for PCSK9-D374Y binding to the high-affinity sites was 125 ± 20 nm Other investigators have reported an apparent Kd 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 Kd estimated here The apparent Kd of PCSK9-S127R binding to the high-affinity LDLR site (548 nm), as measured by its ability to inhibit [125I]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 Kd obtained in a Biacore study [19] The Kd values derived for the lower-affinity class of sites are shown in Table In this context, it is worth mentioning that the existence of high-affinity (32 nm) and low-affinity (86 nm) states has previously been demonstrated for binding of PCSK9-S127R to the ECD of LDLR at pH 7.5 [25] This study also found highaffinity (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 (Kd = nm) at pH 7.5 and with a slightly higher affinity (Kd = 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 Characterization of the binding of PCSK9 to intact cells increasing the association time increased the proportion 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 conformational 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 reaction in which the first binding step, representing binding 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 conversion rates, i.e rate constants governing the conformational change that leads to the second binding step Thus, the rapid phase of PCSK9 association could represent binding of PCSK9 to dimeric receptors that release bound ligand slowly because they convert rapidly The slow phase of association could represent binding to monomeric receptors that release bound ligand rapidly because they convert slowly The proposed model is supported by the finding that a significant 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 dissociation observed in the presence of unlabeled ligand is unclear This phenomenon has often, but not always, been interpreted as indicative of the presence of negative 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 [125I]TC-PCSK9 initially bound and its low affinity for LDLR, rebinding of dissociated [125I]TC-PCSK9 from the bulk solution cannot account for the slowly dissociating component, although rebinding from the FEBS Journal 278 (2011) 2938–2950 ª 2011 The Authors Journal compilation ª 2011 FEBS 2945 Characterization of the binding of PCSK9 to intact cells S A Mousavi et al putative ‘unstirred layer’ surrounding the cells [42] cannot 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 constants of association and dissociation for [125I]TCPCSK9 at 37 °C are similar to values obtained at °C Our preliminary data indicate that HepG2 cells are able to internalize and degrade [125I]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 125I-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 [125I]TC-PCSK9 from the plasma membrane We are currently trying to develop a wash method that can effectively remove cell surface-bound [125I]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) Na125I 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 occurring gain-of-function mutants of PCSK9 that cause severe hypercholesterolemia [1,11,12] Histidine-tagged PCSK9s (wild type, D374Y, and S127R) were produced by transfection of HEK239 cells, and purified from conditioned media as previously described [44] ([125I]PCSK9), which reacts with tyrosines of the protein However, analysis in single-point binding assays showed only 50–60% of the cell-associated [125I]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 overcome the problem of low specific binding, we used labeling by [125I]TC, which reacts with lysines, and we found [125I]TC labeling of PCSK9 to be more suitable for equilibrium 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 binding of [125I]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 contains no tyrosines (or lysines) Purified wild-type PCSK9 was covalently coupled to [125I]TC by the method of Pittman et al [45], with modifications as described previously [46] Briefly, [125I]TC was prepared by reacting TC (6 lL of 10 mm solution in NaCl ⁄ Pi) with Na125I (1.0 mCi) in IodoGen-precoated tubes (Pierce) for 30–40 at room temperature, followed by transfer to a tube containing cyanuric chloride (6 lL of 10 mM solution in acetonitrile) and potassium iodide (6 lL of 0.1 m solution) for The activated [125I]TC adduct was then incubated with wild-type PCSK9 (300–400 lg in 200 lL of carbonate buffer containing 0.5 mm CaCl2, pH 8.9) for 30–45 Unreacted [125I]TC (and free 125I) were removed by gel filtration with Sephadex G25 columns (PD-10; GE Healthcare) equilibrated with NaCl ⁄ Pi containing 0.2 mm CaCl2 [125I]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 · 105 c.p.m lg)1 The quality and integrity of the [125I]TC-labeled proteins were evaluated by SDS ⁄ PAGE followed by EZBlue staining (Pierce) and autoradiography of the gel [125I]TC-PCSK9 is stable, and can be stored at °C for at least weeks For direct iodination, purified PCSK9 (100 lg in 100 lL of NaCl ⁄ Pi containing 0.5 mm CaCl2, pH 7.4) was incubated with Na125I (0.3 mCi) in IodoGen-precoated tubes for 10– 15 at room temperature The reaction was stopped by transfer to a tube containing 0.9 mL of NaCl ⁄ Pi containing 0.5 mm CaCl2 (pH 7.4) Free 125I was removed by gel filtration as described above Goat anti-(rabbit IgG) was radiolabeled with Na125I as described for direct iodination of PCSK9, except that CaCl2 was omitted Cell culture, buffers, and cell treatments Radiolabeling of proteins We initially attempted to investigate the binding of PCSK9 to HepG2 cells using PCSK9 directly labeled with 125I 2946 HepG2 cells (European Collection of Cell Cultures, Porton Down, UK) were routinely cultured in collagen-coated 75-cm2 tissue culture flasks (BD Biosciences, San Diego, FEBS Journal 278 (2011) 2938–2950 ª 2011 The Authors Journal compilation ª 2011 FEBS S A Mousavi et al CA, USA) in MEM supplemented with 200 mm l-glutamine, nonessential amino acids, and 10% fetal bovine serum (referred to as complete growth medium) in a 5% CO2 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 Biosciences, San Diego, CA, USA) After incubation for 22– 24 h at 37 °C in complete growth medium, cells were washed twice with 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 Collagencoated culture plates were used to avoid cell detachment during the relatively long (> 13 h) durations of some experiments All experiments were performed at °C to prevent endocytosis of bound ligand The cells were also kept at °C during the washing steps The standard binding 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 °C Exposure of cells to low pH did not damage the cells during the chase period ( 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 determined with a hemocytometer All experiments were performed 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 preparations and five different preparations of [125I]TC-labeled proteins were used Determination of specific binding of [125I]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 complete growth medium, and then for 26 h in 3% LPDS-containing Opti-MEM In experiments with cells grown in complete growth medium for 48 h, cells were incubated in lipoprotein-free medium for h at 37 °C before the start of the experiments, to allow internalization of cell surfacebound LDL After 50 h of incubation at 37 °C, cells were washed once with mL of DMEM and incubated in the same medium for 15 at °C The medium was then removed, and triplicate wells were incubated with 0.5 mL of incubation medium containing 10 lgỈmL)1 of antibody against LDLR for 90 at °C The cells were then washed, and the amount of bound antibody against LDLR was measured by subsequent binding of 125I-labeled goat anti-(rabbit IgG) (10 lgỈmL)1) A control in which the anti- Characterization of the binding of PCSK9 to intact cells 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 [125I]TC-PCSK9 (5 lgỈmL) or [125I]TC-PCSK9D374Y (1 lgỈmL)1) for h at °C The cells were washed once with mL of cold wash buffer, NaCl ⁄ Pi containing 0.1 mm CaCl2 and 0.5% BSA, and three times with 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 singlepoint binding assays at high initial cell density Binding-inhibition experiments Cells grown in 12-well plates were washed once with mL of DMEM and incubated in the same medium for at least 15 at °C The medium was removed, and cells were incubated with increasing concentrations of unlabeled (serially diluted two-fold) proteins in a total volume of 480 lL at °C for 15 Labeled ligand was added in a small volume (20 lL) and the cells were incubated at °C for h At the end of incubation, the cells were washed once with mL of cold wash buffer and three times with mL of wash buffer without BSA The wash procedure took  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 incubated with 0.5 mL of binding medium containing [125I]TCPCSK9 ( lgỈmL)1, 70 nm) or [125I]TC-PCSK9-D374Y ( lgỈmL)1, 14 nm) 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 h at °C in binding medium containing [125I]TC-PCSK9 ( lgỈmL)1) or [125I]TCPCSK9-D374Y ( lgỈmL)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 mL of cold DMEM and solubilized, and radioactivity was measured as described above Nonspecific binding was FEBS Journal 278 (2011) 2938–2950 ª 2011 The Authors Journal compilation ª 2011 FEBS 2947 Characterization of the binding of PCSK9 to intact cells S A Mousavi et al measured in two parallel wells in the presence of an excess of unlabeled PCSK9-D374Y Radioactivity released from cells following h of dissociation was more than 90% precipitable in 10% trichloroacetic acid, indicating that the majority of bound [125I-TC-PCSK9 (and [125I]TC-PCSK9-D374Y) molecules remained intact and were still on the cell surface Data analysis All binding and kinetic data were fitted by nonlinear regression with prism (GraphPad Software, CA, USA) For inhibition-binding curves, the raw data were analyzed according to one-site-fit and two-site-fit logIC50 models The data were also analyzed according to the Scatchard method [47] Linear regression analyses of binding data gave dissociation constants (Kd), calculated from the reciprocal of the slopes Association data were fitted by either the one-phase exponential association equation Y = Y0 + (plateau ) Y0)*[1 ) exp() Kx)] or the two-phase exponential association equation Y = Y0 + SpanFast*[1 ) exp() KFast* X)] + SpanSlow*[1 ) exp() KSlowX)] Dissociation data were fitted by either the one-phase exponential decay equation Y = (Y0 ) plateau)*exp() KX) + plateau or the two-phase exponential decay equation Y = plateau + SpanFast*exp() KFastX) + SpanSlow*exp() KSlowX) The data were also analyzed by plotting dissociation data according to ln(Bt ⁄ B0) versus time, where B0 is binding at the onset of dissociation, and Bt is the binding remaining at time t Better fits were determined with the F-test Differences were considered to be significant when P was < 0.05 10 Acknowledgements We thank G Griffiths (Department of Molecular Biosciences, University of Oslo) for critical reading of the 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this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 278 (2011) 2938–2950 ª 2011 The Authors Journal compilation ª 2011 FEBS ... half-time for binding to the Characterization of the binding of PCSK9 to intact cells Fig Nonlinear regression and Scatchard analyses of binding- inhibition data (A) Inhibition of [125I]TC-PCSK9 (2.5... 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 [125I]TC-PCSK9 and [125I]TC-PCSK9-D374Y... [125I]TC-PCSK9 binding by unlabeled wild -type PCSK9 (C) Inhibition of [125I]TCPCSK9-D374Y (1 lgỈmL)1) binding to HepG2 cells (9. 2? ?9. 8 · 105) by increasing concentrations of unlabeled PCSK9-D374Y Error