SUPPLEMENTAL INFORMATION
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1 SUPPLEMENTAL INFORMATION EXPERIMENTAL PROCEDURES Tryptic digestion protection experiments - PCSK9 with Ab-3D5 (1:1 molar ratio) in 50 mm Tris, ph 8.0, 150 mm NaCl was incubated overnight at 4 o C. The PCSK9:Ab-3D5 complex was repurified on a S-200 size exclusion column and then digested with trypsin (Promega) in 50 mm Tris, ph 8.0, 50 mm NaCl, ph 8 at an enzyme : substrate ratio of 1 : 1000 (w/w). PCSK9 and Ab-3D5 alone were also digested under the same conditions. Aliquots of the resulting samples collected at different time points were treated with tris(2-carboxyethyl)phosphine (2 mm final concentration) to reduce disulfide bonds and were analyzed in parallel with non-reduced samples. Samples were mixed 1 : 1 with sinapinic acid matrix (100 mm in methanol:acetonitrile:water at a ratio of 56:36:8; Agilent Technologies), applied to the sample stage of a MALDI-TOF/TOF mass spectrometer (4800, Applied Biosystems) and acquired in linear mode with delayed extraction laser shots were averaged for each spectrum acquired. The cleavage sites on PCSK9 and PCSK9:Ab-3D5 complex at 5, 15, 30, 60, 120, 240 min digestions were determined by mapping observed peptide masses onto the sequence of PCSK9 with the program GPMAW (Lighthouse data, v8.0). The identities of selected peptides were confirmed by tandem mass spectrometry on the same instrument. Purification of furin-cleaved PCSK9 - PCSK9 was treated with 80 nm furin in PCSK9 buffer plus 4 mm CaCl 2 for 20 h. Then, Ab-3D5 (IgG) and anti-furin antibody (IgG) (H-220, Santa Cruz Biotechnology) were added to remove intact PCSK9 and furin, respectively. After 30 min incubation the mixtures were applied to a S-200 size exclusion column (HiLoad TM 16/60 Superdex TM prep grade, GE Healthcare) to separate cleaved and intact PCSK9. Biolayer interferometry - For antibody affinity measurements, cleaved PCSK9 forms were produced without Ab-3D5 affinity purification. The same purification protocol described in EXPERIMENTAL PROCEDURES was used, except that Ab-3D5 was not added to the cleaved material. Therefore, these cleaved PCSK9 preparations (designated PCSK9c_hep* and PCSK9c_fu*) still contained residual intact PCSK9. The binding affinities of Ab-3D5 and Ab- 7G7 were determined by biolayer interferometry using an Octet Red384 system (ForteBio). Antibodies (50 µg/ml) were immobilized on anti-murine lgg Fc capture biosensors (ForteBio) in 50 mm Tris, ph 7.5, 300 mm NaCl, 2 mm CaCl 2, 1 mg/ml BSA, 0.1% Tween-20. Measurements of the association and dissociation constants were carried out in the presence of various concentrations of cleaved and intact PCSK9 (0!1 µm). Kinetic parameters k on,, k off and K D were calculated from a nonlinear fit of the data using the Octet software Version 6.1 (ForteBio). Each reported value represents the average ± SD of three experiments. For antibody competition binding experiments LDL receptor ectodomain (R & D Systems) was biotinylated according to manufacturer s instructions (Thermo Scientific). Biotinylated LDL receptor (15!g/ml) was immobilized on a streptavidin biosensor (ForteBio) and immersed into mixtures of 250 nm human PCSK9 pre-incubated for 30 min with 1 µm Ab- 3D5 or Ab-7G7 in 50 mm Tris, ph 7.5, 300 mm NaCl, 2 mm CaCl 2, 1 mg/ml BSA, 0.1% Tween-20. Steady-state binding was measured on an Octet Red384 system (ForteBio) and the results were expressed as percentage of the steady-state binding of human PCSK9 control. The results were the average ± SD of three independent experiments. Cell surface LDL receptor assay with HepG2 cells - HepG2 cells (American Type Culture Collection) were seeded into 48-well plates (Corning) at 1 " 10 5 cells per well in high glucose medium (DMEM, Gibco) containing 2 mm glutamine (Sigma), penicillin/streptomycin (Gibco) and 10% FBS (Sigma) and incubated overnight. Then the medium was changed to DMEM containing 10% lipoprotein deficient serum (LPDS, Intracel). After 24 h, 15 µg/ml PCSK9 was pre-incubated with the indicated concentrations of Ab-7G7 or Ab-3D5 for 30 min 1
2 and added to the cells for 4 h at 37 C. Cells were rinsed with PBS and detached using cell dissociation buffer (Gibco). The cells were collected, centrifuged, and incubated with 1:20 anti-ldl receptor antibody (Progen Biotechnik) in in PBS-1% BSA on ice for 10 min. The samples were then washed with PBS and incubated with 1:200 goat anti-mouse IgG (H + L) Alexa Fluor 488 (Invitrogen) on ice for 5 min. After two PBS washes cells were re-suspended in PBS containing 10 µg/ml of propidium iodide and analyzed on a dual laser flow cytometer (FACScan). Relative fluorescence units were used to quantify LDL receptor expression levels on the HepG2 cell surface. Results were expressed as percent of LDL receptor levels measured in the absence of PCSK9 (=control) and are shown as the average ± SD of three independent experiments. ELISA for measuring PCSK9 binding to LDL receptor - A competition binding ELISA was used to assess the binding affinity of furin-cleaved (PCSK9c_fu) or hepsin-cleaved PCSK9 (PCSK9c_hep) to LDL receptor. Briefly, 1 µg/ml of recombinant human LDL receptor ectodomain (R & D Systems) in 50 mm sodium carbonate, ph 9.6 was immobilized overnight to 384-well MaxiSorp plates (Nalge Nunc International) at 4 C. Then 0.5 µg/ml of biotinylated PCSK9 in 25 mm HEPES, ph 7.2, 150 mm NaCl, 0.2 mm CaCl 2, 0.1% BSA, 0.05% Tween-20 was mixed with an equal volume of serially diluted PCSK9c_fu or PCSK9c_hep and incubated for 30 min at room temperature. The pre-mixed solutions were added to LDL receptor-coated plates and incubated for 2 h at room temperature. The bound biotinylated PCSK9 was detected by sequential additions of streptavidin!horseradish peroxidase (GE Healthcare) and substrate 3, 3#, 5, 5#-tetramethyl benzidine. The mean absorbance values from duplicate wells of three independent experiments were plotted as a function of PCSK9 concentration; the half maximal inhibitory concentration (IC 50 ) values were derived from fitting to a four-parameter equation using KaleidaGraph (Synergy Software).! SUPPLEMENTAL FIGURE LEGENDS SUPPL. FIG. 1. Ab-3D5 epitope mapping. A. Tryptic digestion protection mass spectrometry. Tryptic digests of PCSK9 alone or of PCSK9:Ab-3D5 complexes were analyzed on a MALDI- TOF/TOF mass spectrometer. The sequences of the identified peptides are shown in pink (peptides present in digests of both PCSK9 alone and PCSK9:Ab-3D5 complex) and in blue (peptides not observed in PCSK9:Ab-3D5 complex). The latter peptides all end with Arg215 or with Arg218. The protection of trypsin cleavage by Ab-3D5 at these positions suggests that part of the Ab-3D5 epitope is encompassed by the 218-loop. B. Epitope mapping by overlapping peptide library screening. Overlapping peptides spanning the entire PCSK9 sequence (see Suppl. Fig. 2) were synthesized and binding to Ab-3D5 measured in an ELISA (GenScript, Piscataway NJ). Ab-3D5 only bound to two peptides, peptide #60 and peptide #61. The consensus binding sequence was Glu211-Ala220 (highlighted), which encompasses the 218-loop. The residues Arg215 and Arg218 are boxed in red. SUPPL. FIG. 2. List of peptides. 137 synthesized peptides (GenScript, Piscataway NJ) spanning the entire PCSK9 sequence. Peptide #60 and peptide #61 that showed binding to Ab-3D5 are boxed in red. SUPPL. FIG. 3. Inhibition of PCSK9 function by Ab-3D5. A. Inhibition of PCSK9 binding to LDL receptor by biolayer interferometry. LDL receptor ectodomain was immobilized on the sensor of an Octet Red384 system (ForteBio; Menlo Park, CA) and binding to PCSK9 alone (Ctrl) or PCSK9 preincubated with Ab-3D5 or the non-blocking Ab-7G7 was measured. Results are the average ± SD of three independent experiments. The apparent increased binding of Ab- 2
3 7G7 is due to the increased mass of the PCSK9:Ab-7G7 complex (vs Ctrl = PCSK9 alone) bound to the LDL receptor-loaded sensor tip. B. Ab-3D5 prevents LDL receptor degradation in HepG2 cells. HepG2 cells were treated for 4 h with buffer alone (Ctrl), with PCSK9 alone (-Ab) or with PCSK9 preincubated with increasing concentrations of the non-blocking Ab-7G7 or with Ab- 3D5. Surface LDL receptor levels were quantified by FACS analysis and expressed as percent of control levels. Values are the average ± SD or three experiments. SUPPL. FIG. 4. Purification of furin-cleaved PCSK9 by size exclusion chromatography. PCSK9 was treated with furin (80 nm) for 20 h. After addition of anti-furin IgG and Ab-3D5, proteins were separated on a S-200 size exclusion columns (HiLoad TM 16/60 Superdex TM prep grade, GE Healthcare). The high molecular weight complex of intact PCSK9:Ab-3D5 was separated from the pure furin-cleaved PCSK9 that eluted in later fractions (Figure 4A shows the corresponding SDS-PAGE analysis of elution fractions). SUPPL. FIG. 5. The N-terminal segment makes extensive contacts with PCSK9. The structure of PCSK9 (PDB 3H42) is shown as surface and ribbon representations. The prodomain (blue) and the C-terminal domain (pink) are shown as surface representation. The N-terminal segment of the catalytic domain (Ser153-Arg218) is shown as orange ribbon and the rest of the catalytic domain is in grey surface representation. Inset, the N-terminal segment (orange ribbon) contributes to the "-sheet in the catalytic domain and makes extensive main chain hydrogen bonds with neighboring $-strands (blue dashed lines).! 3
4 Supplemental Table 1. Affinity constants of antibody binding to intact and cleaved PCSK9 Ligand Analyte K D (10-9 x M) Ab-3D5 Ab-3D5 Ab-3D5 PCSK9 PCSK9c_hep* PCSK9c_fu* 3.5 ± 0.3 > ± 29 Ab-7G7 Ab-7G7 Ab-7G7 PCSK9 PCSK9c_hep* PCSK9c_fu* 19.9 ± ± ± 1.7! Kinetic constants were determined by biolayer interferometry using immobilized anti-pcsk9 antibodies Ab-3D5 and Ab-7G7. Furin- and hepsin-cleaved PCSK9 were purified by size exclusion chromatography without using Ab-3D5. Therefore, these cleaved PCSK9 preparations (designated PCSK9c_hep* and PCSK9c_fu*) still contained residual intact PCSK9. Values are the average ± SD of three independent experiments! 4
5 Supplemental Table 2. LDL receptor competition binding ELISA with furin- and hepsin-cleaved PCSK9 Competitor IC 50 ± SD (nm) PCSK9 PCSK9c_hep PCSK9 PCSK9c_fu ± ± ± ± 24.0 Increasing concentrations of purified furin- and hepsin-cleaved PCSK9 (PCSK9c_fu and PCSK9c_hep) were incubated with biotinylated intact PCSK9 (0.5!g/ml) and binding to LDL receptor immobilized on 384-well MaxiSorp plates was determined. The halfmaximal inhibitory concentrations (IC 50 values) were calculated by fitting the data to a four-parameter equation. The values are the average ± SD of three independent experiments. 5
6 A Furin cleavage site SIPWNLERITPPRYRADEYQPPDGGSLVEVYLLDTSIQSDHREIEGRVMVTDFENVPEEDGTRFHRQASKCDSHGTHLAGVVSGRDAGVAKGASMRSLRVLNCQGK SIPWNLER YRADEYQPPDGGSLVEVYLLDTSIQSDHREIEGR ADEYQPPDGGSLVEVYLLDTSIQSDHREIEGR SIPWNLERITPPRYRADEYQPPDGGSLVEVYLLDTSIQSDHREIEGRVMVTDFENVPEEDGTRFHR SIPWNLERITPPRYRADEYQPPDGGSLVEVYLLDTSIQSDHREIEGRVMVTDFENVPEEDGTR YRADEYQPPDGGSLVEVYLLDTSIQSDHREIEGRVMVTDFENVPEEDGTRFHR YRADEYQPPDGGSLVEVYLLDTSIQSDHREIEGRVMVTDFENVPEEDGTR ADEYQPPDGGSLVEVYLLDTSIQSDHREIEGRVMVTDFENVPEEDGTRFHR ADEYQPPDGGSLVEVYLLDTSIQSDHREIEGRVMVTDFENVPEEDGTR VMVTDFENVPEEDGTRFHR VMVTDFENVPEEDGTR GASMRSLRVLNCQGK B PCSK9 residue Peptide #60 V P E E D G T R F H R Q A Peptide #61 E D G T R F H R Q A S K S 6 Supplemental Figure 1
7 7 Supplemental Figure 2
8 A PCSK9 binding to LDLR (%) Ctrl 7G7 3D5 B Cell surface LDLR (% of ctrl) Ctrl -Ab Ab-7G7 ( M) Ab-3D5 ( M) Supplemental Figure 3 8
9 kDa 158kDa 44kDa cleaved PCSK9 mau 150 intact PCSK9 + Ab-3D Elution volume (ml) Supplemental Figure 4 9
10 Catalytic domain S153 R218 C-terminal domain Prodomain 10 Supplemental Figure 5
Nature Methods: doi: /nmeth Supplementary Figure 1
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