Mutagenesis of PCSK9
D374Y-PCSK9-FLAG plasmid containing mutation D374Y was made by site-directed mutagenesis of the WT PCSK9 plasmid (pCMV-PCSK9-FLAG) which contains the sequence for the FLAG epitope tag fused to the 3' end of the PCSK9 coding sequence, as previously described . To create the D374Y-PCSK9-His plasmid, D374Y-PCSK9 was first amplified using the D374Y-PCSK9-FLAG plasmid as a template and the 5' primer: 5'-CACCATGGGCACCGTCAGCTCCAG-3' and 3' primer: 5'-CTGGAGCTCCTGGGAGGCCTGCGC-3'. The PCR product was cloned into pcDNA3.1D/V5-His-TOPO with pcDNA3.1 Directional TOPO Expression Kit (Invitrogen, Carlsbad, CA). The resulting fusion protein (D374Y-PCSK9-His) contains D374Y-PCSK9 fused to a 47 amino acid peptide containing the V5 and His epitope tags. The WT-PCSK9-His plasmid was created by site-directed mutagenesis of the D374Y-PCSK9-His plasmid using the 5' primer: 5'-CATTGGTGCCTCCAGCGACTGCAGCACCTGC-3' and 3' primer: 5'-GCAGGTGCTGCAGTCGCTGGAGGCACCAATG-3'. An expression vector that encodes WT- PCSK9 followed by a red fluorescent protein was constructed as follows. The WT-PCSK9-FLAG plasmid was used as template for amplification of WT-PCSK9 using the primers: 5' primer; 5'-CCCTCGAGATGGGCACCGTCAGCTCCAGGCGG-3', 3' primer; 5'-ATCCCGGGCCTGGAGCTCCTGGGAGGCCTGCGCCA-3'. The 5' primer contained a XhoI restriction site, and the 24 first nucleotide of the translated part of the PCSK9 gene. The 3' primer contained a SmaI site and the last 27 nucleotides of the translated part of the PCSK9 gene. The stop codon was not included in the 3'-primer. The amplified PCR product was digested with XhoI and SmaI (New England Biolabs Inc., Ipswich, MA). The digested products were separated by agarose gel electrophoresis. The relevant band was excised and purified prior ligation into a similarly digested pDsRed-Express-N1 plasmid (Clontech Laboratories, Inc., Palo Alto, CA) to generate WT-PCSK9-Red. The integrity of the plasmids was confirmed by DNA sequencing. An empty plasmid, pcDNA3.1/c-myc-His (Invitrogen), was used as a negative control in transfection experiments.
Production of conditioned medium from transiently transfected HepG2 cells
HepG2 cells (The European Collection of Cultured Cells, Wiltshire, UK) were cultured in Modified Eagle's medium (MEM) (Gibco, Carlsbad, CA), containing penicillin (50 U/ml), streptomycin (50 μg/ml), L-glutamine (2 mM) and 10% fetal calf serum (Invitrogen) in a humidified atmosphere (37°C, 5% CO2). For transient transfection experiments, HepG2 cells were seeded in 75 cm2 collagen-coated flasks (BD Biosciences, San Diego, CA) and grown to 80% confluency. The cells were transiently transfected with PCSK9-containing plasmids or with empty plasmid using Fugene-6 Reagent (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's instructions. A ratio of 6:1 reagent to plasmid DNA was used during transfections. Transfection efficiencies were in the range of 10–20%. 24 h after transfection, the transfection solution was removed and the cells were washed once with phosphate-buffered saline (PBS) before addition of 15 ml serum-free OptiMEM medium (Gibco). After a further 24 h incubation, the medium was removed and centrifuged for 5 min at 3000 rpm. This medium is referred to as conditioned medium. Constructs with a His sequence tag were used to generate PCSK9-His fusion proteins in order to enable immobilization of PCSK9-His on a nickel-chelating resin. Conditioned media from cells transfected with PCSK9-His constructs were also used for incubations with membrane fractions and with a preparation of truncated LDLR lacking the cytoplasmic and transmembrane domains as well as the experiments involving hypertonic media. For the other experiments, PCSK9 constructs with a FLAG sequence tag were used. The LDLR-degrading activity of conditioned medium from HepG2 cells transiently transfected with D374Y-PCSK9-FLAG plasmid, was maintained for at least two weeks when stored at 4°C (data not shown).
Western blot analysis
Cells were lysed in a buffer containing 1% Triton X-100, 100 mM NaCl, 10 mM EDTA, 20 mM Tris-HCl (pH 7.5). Cell lysates equivalent to 20 μg protein, or membrane fractions equivalent to 10 or 15 μg were separated by gel electrophoresis using a 4–20% Tris-HCl Criterion Precast Gel (Bio-Rad, Hercules, CA). The proteins were then electrophoretically transferred to an Immun-Blot PVDF Membrane for Protein Blotting (Bio-Rad). Non-specific binding sites were blocked in 5% Blotting Grade Blocker Non-Fat Dry Milk (Bio-Rad) for 1 h or overnight, and the membrane was immunostained for 1 h. The antibodies used were rabbit IgG anti-LDLR (1:1000, Progen Biotechnik GmbH, Heidelberg, Germany), rabbit IgG anti-PCSK9 (1:200, Cayman Chemical Company, Ann Arbor, MI), mouse anti-human transferrin receptor (1:1000, Zymed, San Fransisco, CA), mouse IgG anti-human CD71 (1:1000, Nordic Biosite, Täby, Sweden) and mouse anti-FLAG M2 monoclonal antibody (1:3000, Sigma-Aldrich Corp., St. Louis, MO). After incubation with the primary antibody the membranes were washed twice in Tris-buffered saline (TBS) containing 0.1% Tween-20 (Sigma-Aldrich Corp.) and incubated for 1 h with sheep anti-rabbit IgG or sheep anti-mouse IgG (Amersham Biosciences, Little Calfont, UK), both conjugated with horseradish peroxidase. After two more washing steps with TBS containing 0.1% Tween-20, the bands were visualized with SuperSignal West Dura Extended Duration Substrate (Pierce Biotechnology, Rockford, IL) and chemiluminescence was detected on a ChemiDoc XRS (Bio-Rad). Quantity One version 4.4.0 software (Bio-Rad) was used for quantification of band intensities.
Flow cytometric analysis of internalization of LDL and cell surface LDLR
A FACS Canto flow cytometer (BD Biosciences) was used to determine the amounts of LDL internalized by cultured cells and the amounts of cell surface LDLR, as previously described . Briefly, LDL was fluorescently labelled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate (DiD) (Molecular Probes, Eugene, OR), added to the cells (final conc. 10 μg/ml) and incubated for 2 h at 37°C. Cell surface LDLR were labelled with anti-LDLR IgG-C7 mouse monoclonal antibody (1:20, Progen Biotechnik) and counter-stained with Alexa Fluor® 488 goat anti-mouse IgG (H+L) (1:400, Molecular Probes). The amount of LDL internalized and cell surface LDLR were then determined by flow cytometry. LDLR levels were corrected for unspecific staining by labelling the cells with secondary antibody only.
Gel filtration of conditioned medium
Conditioned medium was concentrated approximately 10 times using BJP concentrators (Pro-Chem Inc., Littleton, MA) and 1 ml was loaded on a Superdex 200 gel filtration column (Pharmacia Fine Chemicals, Uppsala, Sweden). PBS was used for elution at a flow rate of 1 ml/min and fractions of 2 ml were collected. Gel Filtration Standard (Bio-Rad) containing five different proteins with molecular weights ranging from 1.35 to 670 kDa was used together with ferritin (440 kDa) for size determination. To study the ability of the fractions to reduce the internalization of LDL, HepG2 cells were incubated with the different fractions for 3 h at 37°C. Fluorescently labelled LDL was then added and the cells were incubated for another 2 h at 37°C. The amounts of LDL internalized were determined by flow cytometry.
Preparation of a truncated LDLR lacking the cytoplasmic and membrane-spanning domains
An LDLR construct encoding the 21 amino acids of the signal peptide, the 767 amino acids extracellular domain and the first amino acid of the membrane-spanning domain, was constructed by mutagenesis of the plasmid pcDNA4-LDLR-EYFP  which contains the coding sequence of the LDLR. This construct encodes the extracellular (EC) part of the LDLR. The primers used to amplify the truncated LDLR were: 5' primer: 5'-CTTGGTACCAGCATGGGGCCCTGGGGCTGGAAATTGC-3', 3' primer: 5'-CGAGCGGCCGCAGCCCTCACGCTACTGGGCTTCTTC. The 5' primer contained a KpnI restriction site, a Kozak sequence and the first 25 nucleotides of the translated part of the LDLR cDNA. The 3' primer contained a NotI site and nucleotides 2343–2467 of the LDLR cDNA. The amplified PCR product was TA-cloned into the pCR® 2.1-TOPO vector of the TOPO TA cloning kit (Invitrogen) according to the instructions by the manufacturer. The resulting pCR® 2.1-EC-LDLR plasmid was digested with KpnI and NotI (new England Bioloabs, Inc.), and the fragments were separated by agarose gel electrophoresis. The band corresponding to EC-LDLR was cut out of the gel and purified by QIAquick gel extraction kit (Qiagen, Hilden, Germany) prior to ligation into a similarly digested pcDNA3.1/c-myc-His plasmid (Invitrogen) to generate pcDNA3.1-EC-LDLR-His. This plasmid contains the truncated LDLR cDNA fused to the c-myc and His sequence tags. The integrity of the plasmid was verified by DNA sequencing. The truncated LDLR (EC-LDLR-His) without the cytoplasmic and membrane-spanning domains, was prepared from media of HepG2 cells transiently transfected with the pcDNA3.1-EC-LDLR-His plasmid. 5 ml of medium containing secreted EC-LDLR-His was concentrated to 200 μl using BJP protein concentators (Pro-Chem Inc.). 10 μl of this concentrated medium was then added to 50 μl conditioned medium containing either WT-PCSK9-His or D374Y-PCSK9-His. Conditioned medium from HepG2 cells transfected with empty plasmid was used as a control. After incubation for 3 h at 37°C, 20 μl was subjected to western blot analysis in order to quantify the amount of EC-LDLR-His.
Cell membrane preparations
HepG2 cells were cultured in serum-free OptiMEM medium overnight and harvested by scraping. After sonication, the cells were centrifuged at 500 g for 10 min. The supernatant was removed and centrifuged at 100 000 g for 60 min in a Beckman Coulter Optima LE-80K ultracentrifuge (Beckman Coulter, Fullerton, CA) using an ultrarotor 70TI (Beckman Coulter). The pellet containing the cell membranes was dissolved in PBS. 25 μl of cell membranes was incubated with 25 μl conditioned medium from HepG2 cells transiently transfected with empty plasmid, WT-PCSK9-His plasmid or D374Y-PCSK9-His plasmid.
Affinity chromatography of His-tagged fusion proteins
WT-PCSK9-His or D374Y-PCSK9-His secreted from HepG2 cells transiently transfected with WT-PCSK9-His or D374Y-PCSK9-His plasmids were isolated from 2 ml of culture medium added 500 μl 5× Native Purification Buffer with imidazole (final conc. 30 mM) using the ProBond Purification System (Invitrogen) according to the manufacturer's instructions. The Resin-Purification Column contains Nickel-Chelating Resin which selectively binds His-tagged recombinant fusion proteins. The cleared medium was sequentially dialyzed against PBS and OptiMEM serum-free medium at 4°C using a Slide-A-Lyzer Dialysis Cassette (10 000 MWCO, Pierce Biotechnology).
LDLR in hypertonic medium
Hypertonic medium was prepared by a slight modification of the method described by Krämer-Guth et al. . 30 μl of 5.0 M NaCl was added per ml of OptiMEM medium to increase he final concentration of NaCl by 150 mmol/l. The hypertonic medium was added to untransfected HepG2 cells for a 30 min preincubation at 37°C. The hypertonic medium was replaced with conditioned medium from transiently transfected HepG2 cells which had been made hypertonic after it had been removed from the transfected cells. The amount of cell surface LDLR in untransfected HepG2 incubated with hypertonic, conditioned medium, was determined by flow cytometry as previously described.
Degradation of the LDLR in the presence of nocodazole or ammonium chloride
HepG2 cells were seeded in 25 cm2 flasks (106 cells/flask) and cultured overnight. Nocodazole (Sigma-Aldrich Corp., final conc. 20 μg/ml) or ammonium chloride (Sigma-Aldrich Corp., final conc. 10 mM) was added to the cells and incubated for 30 min at 37°C. Medium was then replaced with conditioned medium containing nocodazole or ammonium chloride (final conc. 20 μg/ml and 10 mM, respectively). After incubation (3 h, 37°C), the cells were washed twice in PBS and harvested by cell scraping. Cell membranes were isolated with Compartmental Protein Extraction Kit (Chemicon International, Temecula CA) according to the manufacturer's instructions. Yields were typically 60–100 μg of membrane proteins per flask of which 10 μg were used for western blot analysis.
Comparisons between groups were performed by Student's two-tailed t-test. Values are reported as mean (± SD).