Antibody-mediated disruption of the interaction between PCSK9 and the low-density lipoprotein receptor - PubMed (original) (raw)
Antibody-mediated disruption of the interaction between PCSK9 and the low-density lipoprotein receptor
Christopher J Duff et al. Biochem J. 2009.
Abstract
PCSK9 (proprotein convertase subtilisin/kexin type 9) promotes degradation of the LDLR [LDL (low-density lipoprotein) receptor] through an as-yet-undefined mechanism, leading to a reduction in cellular LDLc (LDL-cholesterol) and a concomitant increase in serum LDLc. Central to the function of PCSK9 is a direct protein-protein interaction formed with the LDLR. In the present study, we investigated a strategy to modulate LDL uptake by blocking this interaction using specific antibodies directed against PCSK9. Studies using surface plasmon resonance demonstrated that direct binding of PCSK9 to the LDLR could be abolished with three different anti-PCSK9 antibodies. Two of these antibodies were raised against peptide epitopes in a region of the catalytic domain of PCSK9 that is involved in the interaction with the LDLR. Such antibodies restored LDL uptake in HepG2 cells treated with exogenous PCSK9 and in HepG2 cells engineered to overexpress recombinant PCSK9. This latter observation indicates that antibodies blocking the PCSK9-LDLR interaction can inhibit the action of PCSK9 produced endogenously in a cell-based system. These antibodies also disrupted the higher-affinity interaction between the natural gain-of-function mutant of PCSK9, D374Y, and the LDLR in both the cell-free and cell-based assays. These data indicate that antibodies targeting PCSK9 can reverse the PCSK9-mediated modulation of cell-surface LDLRs.
Figures
Figure 1. Binding affinity of wild-type PCSK9 and the D374Y mutant for the LDLR
(A) FLAG-tagged versions of wild-type (WT) PCSK9 and the D374Y mutant were purified as described in the Experimental section, and 1 μg of each protein was subjected to electrophoresis on a 4–12% acrylamide NuPAGE Novex gel (Invitrogen) followed by staining with Coomassie Blue. Molecular-mass markers (sizes in kDa) are indicated on the left. The purified extracellular domain of the LDLR was immobilized on the surface of a CM5 sensorchip, and wild-type PCSK9 (B) and the D374Y mutant (C) at concentrations of 7000, 1750, 438, 109, 27, 7, 2 and 0 nM were passed over the surface as described in the Experimental section. The response from the reference surface was subtracted, and the baselines adjusted to zero to give response over time binding curves.
Figure 2. Antibody interference of the PCSK9–LDLR interaction
LDLR was immobilized via amine coupling to a CM5 sensorchip. Wild-type PCSK9 at 50 nM was pre-incubated for 1 h at 4 °C with one of four anti-PCSK9 polyclonal antibodies (AF3888, E6675, E6660 or Ab28770) or a non-specific control antibody (anti-IL1Ra) and passed over the sensorchip surface as described in the Experimental section. (A) Representative sensorgram showing the significantly decreased binding level observed in the presence of 500 nM antibody AF3888. (B) Relative binding level observed in the presence of 500 nM of the indicated antibodies. Results are the means±S.E.M. for three independent experiments. *P<0.05 compared with no antibody.
Figure 3. Predicted epitopes of the antibodies on the surface of PCSK9
(A) The epitopes of the anti-peptide antibodies E6675 (orange) and E6660 (magenta) were mapped on to the surface of the crystal structure of PCSK9 (PDB code 2P4E). (B) The epitope for Ab28770 (red) and the region shown to interact with the EGF-A domain of the LDLR (blue) mapped on to the structure of PCSK9. The prodomain is shown in grey, the catalytic domain is shown in green, the CHRD is shown in yellow, and the site of the gain-of-function mutation D374Y is shown in black. Figure drawn using PyMOL (DeLano Scientific;
).
Figure 4. Blocking the extracellular PCSK9–LDLR interaction inhibits PCSK9-mediated LDLR degradation and restores LDL uptake
(A) HepG2 cells were incubated for 6 h with various concentrations of purified PCSK9 [wild-type (WT) or D374Y] in the presence of BODIPY®–LDL, the cells were then washed twice with PBS, and the fluorescence was measured at 485 nm excitation/535 nm emission. Results are presented as a percentage of the LDL taken up by the cells in the absence of PCSK9. Purified wild-type PCSK9 (B) or the D374Y mutant (C) at 2.5 μg/ml (34.7 nM) were pre-incubated for 1 h with increasing concentrations of antibody AF3888 or control antibodies IL1Ra and Ab28770 before addition to the HepG2 cells and determination of their relative LDL uptake. Results are the percentage reversal of the PCSK9 effect on LDL uptake, and are means±S.E.M. for three independent experiments each performed in triplicate. *P<0.05.
Figure 5. Antibody AF3888 counteracts the effect of PCSK9 expression on LDLR degradation in HepG2 cells
(A) HepG2 cells were transduced with recombinant BacMam virus constructs encoding wild-type (WT) PCSK9, D374Y or RRRR218EL as described in the Experimental section. After 42 h, the medium was changed and replaced with serum-free growth medium containing 10 μg/ml BODIPY®–LDL. After 6 h, the cells were washed twice with PBS and the fluorescence was measured at 485 nm excitation/535 nm emission. Results are presented as a percentage of the LDL taken up by untransduced cells and are representative of three independent experiments, each carried out in triplicate. *P<0.05. (B) Western blot of secreted PCSK9 (wild-type, D374Y and RRRR218EL) in the medium of HepG2 cells after BacMam-mediated overexpression. PCSK9 was detected via its FLAG tag using an anti-FLAG M2 peroxidase-conjugated antibody. For antibody-blocking experiments, immediately after transduction with either wild-type (WT) PCSK9 (C) or D374Y (D) BacMam virus, the inoculum was replaced with normal growth medium containing various concentrations of antibody AF3888 or control antibody IL1Ra. After 24 h, the medium was replaced with fresh medium containing antibody at the same concentration. After a further 16 h, the medium/antibody mixture was replenished, this time with the addition of 10 μg/ml BODIPY®–LDL. After 6 h, the cells were washed twice with PBS and their relative fluorescence was determined. Results are presented as percentage reversal of the PCSK9 effect on LDL uptake. Cell viability (determined using the Cell Titre-Glo® kit from Promega) is presented as a percentage of control (BacMam-transduced, antibody-untreated cells) after antibody-blocking experiments for wild-type PCSK9 (E) and D374Y (F) transductions. For (C–F), black bars indicate AF3888, and white bars indicate IL1Ra. Results are the means±S.E.M. for three independent experiments each performed in triplicate, except for (D) which are the mean±S.E.M. for three independent experiments each performed in duplicate. *P<0.05.
References
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