The plasmid pFC14A-PCSK9 was produced in three steps. First, SgfI and PmeI endonuclease sites were introduced into the 5'-end or 3'-end of the human PCSK9 gene, respectively, by PCR using the oligonucleotides 5'- CA-CGGCGATCGCCATGGGCACCGTCAGCTCCAGGC-3' (5' primer) and 5'-CTTGGTTTAAACCTGGAGCTCCT-GGGAGGCCTGC-3' (3' primer), and pCDNA3.1/V5-His-PCSK9 [22Fisher TS, Lo Surdo P, Pandit S, et al. Effects of pH and low density lipoprotein (LDL) on PCSK9-dependent LDL receptor regulation J Biol Chem 2007; 282: 20502-12.] as a template. Next, the amplified PCSK9 ORF was sequenced and cloned into the pF4K CMV Flexi Vector (Promega, WI, USA) following the standard cloning procedure [23Russell DW, Sambrook J. Molecular cloning: a laboratory manual 2001.]. The resulting plasmid was sequenced and cloned into the pFC14A HaloTag CMV Flexi Vector (Promega) to produce pFC14A-PCSK9. Prior to transfection, pFC14A-PCSK9 and pCDNA3.1/hygro/lacZ (Invitrogen, NY, USA) were linearized with BglII (New England Biolabs, MA, USA). Huh7 cells were seeded on day 0 at 1.5×10⁶ cells in 60 mm dishes and cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS, v/v, Invitrogen) at 37°C and 5% CO₂ overnight. On day 1, the cells were co-transfected with linearized pFC14A-PCSK9 and pCDNA3.1/hygro/lacZ using GenJet In vitro DNA transfection reagent for Huh7 cells (Ver. II) (SignaGen Laboratories, MD, USA) following the manufacturer's instructions. At 48 h post transfection (day 3), the cells were fed with selective media (DMEM supplemented with 10% FBS and 200 µg/ml Hygromycin B [Invitrogen]), and re-plated onto 100 mm dishes. On day 17, cells growing in selective media were collected and labeled with HaloTag R110Direct Ligand (Promega) according to the manufacturer's instructions. The cells expressing HaloTag were sorted using Fluorescence-Activated Cell Sorting (FACS) and 8×10⁴ cells were seeded into 6-well culture plates containing selective media. Cells were allowed to grow and expanded at 37°C and 5% CO₂ for two weeks. On day 31, cells underwent a second round of FACS. The cells were harvested and labeled as described above. Fluorescence positive cells were seeded into 96-well culture plates at 1 cell/well, and cultured in selective media at 37°C and 5% CO₂ until colonies formed. A screen for positive clones was performed as follows: 50 µl of media was transferred from wells containing cell clones to a 96-well assay plate. 1 µl of 0.1 mM HaloTag TMR Ligand (Promega) was added to each well. The plate was incubated at room temperature for 15 min in the dark before adding 10 µl of 6xSDS reducing sample buffer (Boston BioProducts, MA, USA) to each well. Next, 10 µl each of denatured media was loaded onto a 4-12% Bis-Tris SDS-PAGE gel (Invitrogen) and run at 200V for 60 min. The gel was rinsed briefly using Milli-Q H₂O followed by image scanning using a Typhoon (GE Healthcare, NJ, USA) at TMR/Cy3 fluorescence setting. Clones showing the PCSK9-HaloTag band on the gel were selected and expanded. To mimic the physiological PCSK9 level, a stable cell line with medium-low PCSK9-HaloTag expression level, known as 14-13-E4, was established. All the experiments described herein were performed using 14-13-E4.

All antibodies were purchased from external vendors: Goat polyclonal antibody against PCSK9 (Abcam, MA, USA), goat polyclonal antibody against LDL receptor (R&D Systems Inc., MN, USA), mouse monoclonal antibody against PDI (Abcam), mouse monoclonal antibody against Golgin97 (Invitrogen), rabbit polyclonal antibody against EEA1 (Abcam), and mouse monoclonal antibody against LAMP1 (Abcam). The following antibodies were used as isotype controls: mouse IgG1 (BD Biosciences, CA, USA), rabbit IgG (Invitrogen) and goat IgG (Invitrogen). Secondary antibodies conjugated with Alexa Fluor were purchased from Invitrogen including: Alexa Fluor 647 donkey-anti-rabbit IgG, Alexa Fluor 647 donkey-anti-mouse IgG, Alexa Fluor 647 donkey-anti-goat IgG, Alexa Fluor 488 donkey-anti-goat IgG, Alexa Fluor 568 donkey-anti-mouse IgG, and Alexa Fluor 568 donkey-anti-rabbit IgG.

For HaloTag ligand labeling, the HaloTag TMR Ligand and Alexa Fluor 488 Ligand (Promega) were used to label the cells expressing PCSK9-HT following the manufacturer's instructions with some modifications. In brief, clone 14-13-E4 or wild type Huh7 cells were plated on 96-well glass bottom culture plates (Matrical Bioscience, WA, USA) and incubated overnight at 37°C and 5% CO₂ prior to labeling, with the exception of the experiments where overnight labeling of Alexa Fluor 488 was required. In those experiments, 0.2 µM of Alexa Fluor 488 Ligand was added at the time of cell plating. In the other experiments, 0.2 µM of Alexa Fluor 488 Ligand or 5 µM of TMR ligand was incubated with cells in the DMEM media supplemented with 10% FBS on the following day. The incubation time ranged from 30 to 300 min as required in different experiments for Alexa Fluor 488 and 15 min for TMR. The unbound ligand was washed off with DMEM after labeling. For live cell imaging, the media was replaced with phenol-red free DMEM prior to capturing images. For fixed cell imaging, the labeled cells were fixed with 4% para-formaldehyde containing 0.2 M sucrose in PBS for 10 min at room temperature followed by permeabilization with 0.1% Triton X-100 (in PBS, Boston BioProducts). The cells were then washed in DPBS (no Ca²⁺ or Mg²⁺, Invitrogen) three times and incubated overnight with 2% Normal Donkey Serum (Jackson ImmunoResearch Laboratories, PA, USA) and 0.01% Triton X-100 in PBS at 4°C. The cells were incubated for 2 h at room temperature with primary antibodies and then washed three times with DPBS. The corresponding species-specific Alexa Fluor (488, 568, or 647) conjugated secondary antibodies were used to reveal antigen-antibody complexes by incubation for 45 min at room temperature. After three washes in DPBS, the cell nuclei were counterstained with Hoechst 33342 (Invitrogen). Fluorescence was visualized using either a Zeiss LSM 510 confocal microscope equipped with multiple lasers or an Evotech Opera Confocal High Content Imager. Potentially overlapping emissions were collected separately using sequential scanning. On the Zeiss LSM 510, 4 color images (Hoechst, Alexa Fluor 488, Alexa Fluor 568 or TMR, Alexa Fluor 647) were acquired in a two pass configuration. The configuration of pass 1 was as follows: simultaneous 2P (780 nM)/543 laser excitation and 430BP/585BP emission filters plus DIC for transmitted light. The configuration of pass 2 was: 488/633 laser excitation and 515BP/670LP emission filters. Three color images (Alexa Fluor 488, TMR, DRAQ5) were acquired on the Opera as 2 pass images using the following configuration: 488/633 laser excitation 535BP/690BP emission then 568 laser excitation 600BP emission. We also obtained four color images (Hoechst, Alexa Fluor 488, Alexa Fluor 568 or TMR, Alexa Fluor 647) acquired as 2 pass images with the following configuration: 488/633 excitation 535BP/690BP emission, then UV (ARC lamp)/568 laser excitation 600BP emission. All time-lapse and kinetic studies were performed on the Opera using Perkin-Elmer's RCI software.

In the initial step of purification of PCSK9-HT, 800 mL of 14-13-E4 media (lacking phenol red and containing 5% FBS) was applied to a 6 mL Resource Q column (GE Healthcare). The column containing media was washed with seven column volumes of buffer A (25 mM HEPES pH 7.9, 30 mM Sodium Chloride, 0.1 mM Calcium Chloride, 5.0% Glycerol). A salt gradient towards a final of 50% Buffer B (25 mM HEPES pH 7.9, 1.0 M Sodium Chloride, 0.1 mM Calcium Chloride, 5.0% Glycerol) over 20 column volumes was applied for elution. Samples from the peak were run onto 10-20% Tris-Glycine gels and probed with goat polyclonal antibody against PCSK9 (Abcam). The western blot bands were detected with IRDye 800CW donkey-anti-goat IgG (Licor, NE, USA) and scanned with a Licor Odyssey. Positive samples containing PCSK9-HT (~ 270 mM – 350 mM Sodium Chloride) were pooled and concentrated. The concentrated sample was applied to a Superdex 200 10/300GL column (GE Healthcare) and run in buffer containing 25 mM HEPES pH 8.0, 150 mM Sodium Chloride, 0.1 mM Calcium Chloride and 10% glycerol. Samples from the size exclusion chromatography peak were verified by western blot, as noted above, and with Gelcode Blue (Thermo Fisher Scientific, IL, USA) staining. The samples were quantitated using a NanoDrop spectrophotometer and subjected to a LDL uptake assay.

The assay was performed as previously described [22Fisher TS, Lo Surdo P, Pandit S, et al. Effects of pH and low density lipoprotein (LDL) on PCSK9-dependent LDL receptor regulation J Biol Chem 2007; 282: 20502-12.] with some modifications. Parental HepG2 cells were seeded into poly-D-lysine coated 96-well plates Corning, NY, USA) at 30, 000 cells/well in DMEM supplemented with 10% FBS (HyClone, UT, USA) in 100 µl volume. After 24 h of incubation at 37°C and 5% CO₂, the medium was replaced with 100 µl of DMEM lacking serum and the cells were incubated for additional 24 h. On day 3, the medium was aspirated and 100 µl of Assay Mixture [DMEM + 10% LPDS (Intracel, MD, USA) + 10 µg/ml Alexa Fluor-546 (Invitrogen) labeled human LDL (Intracel) + purified PCSK9 in various concentrations] was applied to the cells. The plates were incubated at 37°C and 5% CO₂ for 5 h before being washed with 1xTBS (Bio-Rad, CA, USA) and lysed with 100 µl RIPA buffer (TekNova, CA, USA). The lysate was then transferred to a MicroFluor2 Black “U” bottom plate (Thermo Fisher Scientific). The fluorescent LDL signal was measured using an excitation wavelength of 520 nm and an emission wavelength of 580 nm.

The western blots from cell lysates indicated that the expression level of PCSK9-HT is close to the endogenous level of PCSK9 (~ 2 fold) (Fig. 1A). To investigate whether introduction of the HaloTag to the C-terminal end of PCSK9 would have an impact on PCSK9 function, PCSK9-HT protein was purified from the culture media of 14-13-E4. The partially purified PCSK9-HT was subjected to the LDL uptake assay. As shown in Fig. (1B), the PCSK9-HT effectively inhibited the LDL receptor mediated LDL uptake in HepG2 cells, confirming that the fusion of the HaloTag to the C-terminal of PCSK9 did not affect the function of PCSK9. The potency of PCSK9-HT was lower when compared to the non-tagged wild type recombinant PCSK9. This effect may be the results of the protein's partial purity.

Fig. (1) PCSK9-HT is labeled with HaloTag ligands TMR and Alexa Fluor 488. A Huh7 stable cell line expressing C-terminal PCSK9-HaloTag fusion protein was established and verified with western blot (A, clone 14-13-E4). The expression level of PCSK9-HT is about 2-fold of the endogenous PCSK9 level. B. Purified PCSK9-HT inhibited LDL uptake in a dose dependent manner in HepG2 cells. The de-creased potency of PCSK9-HT (red) compared to wt PCSK9 (blue) is likely due to protein impurity. C. 14-13-E4 cells were labeled with 0.2 µM Alexa Fluor 488 overnight, followed by TMR labeling (5 µM) for 15 min. The unbound ligand was washed off before the second label-ing or image capture. Confocal images were taken with a Zeiss LSM 510 confocal microscope with appropriate fluorescence filters. Intracel-lular PCSK9 (red) and internalized PCSK9 (green) showed different staining patterns.

Two main features of HaloTag ligands are their spectral differences and their different cell permeability. We utilized these features by first labeling the 14-13-E4 cells overnight with the impermeable ligand, Alexa Fluor 488, to bind all the secreted PCSK9 in the cell media. The same group of cells was then labeled with cell permeable ligand, TMR, for 15 min. Through the sequential labeling, we were able to differentiate the two pools of PCSK9: the newly synthesized intracellular pool and the internalized pool following secretion (Fig. 1C). The two pools of PCSK9 have shown a distinct pattern of staining. The PCSK9 staining of intracellular pool was predominantly uniform whereas the internalized pool displayed a characteristic punctate staining. The ability to separate the two pools of PCSK9 utilizing different HaloTag ligands enabled us to further study the trafficking and fate of PCSK9 in 14-13-E4 cells.

Next, we investigated the trafficking patterns of two pools of PCSK9 in 14-13-E4 cells. The experimental design is shown in Fig. (2). To monitor the trafficking of the intracellular pool of PCSK9, 14-13-E4 cells were labeled with 5 mM TMR for 15 min. To allow protein trafficking, the unbound ligand was washed off and the cells were incubated in fresh DMEM (supplemented with 10% FBS) at 37°C + 5% CO₂ from 30 to 300 min. The process was stopped by fixation and permeabilization of cells at various time points. The localization of PCSK9 was determined by counterstaining the cells with different subcellular organelle markers, including PDI (ER), Golgin97 (Golgi complex), EEA1 (early endosome), and LAMP1 (lysosome). As shown in Fig. (3), intracellular PCSK9 was primarily located in the ER and Golgi after 30 min of labeling and subsequently moved mostly to Golgi locations (up to 180 min post-labeling). The localization of PCSK9 was eventually observed in lysosomes (180-300 min post-labeling). Localization of intracellular PCSK9 with early endosomes, noted at 240 min and 300 min of incubation, was observed as well. These endosome/lysosome localized TMR labeled PCSK9 molecules may have transferred to the endosome/lysosome directly from the Golgi, or these proteins may have been secreted out before re-internalized during incubation period.

Fig. (2) Experimental design. Huh7 cells were plated and transfected with the PCSK9-HaloTag fusion gene to establish a stable cell line expressing PCSK9-HT. To label secreted PCSK9-HT, 0.2 µM of Alexa Fluor 488 ligand was incubated with cells in DMEM media supplemented with 10% FBS for various times, from 30 min, 60 min, 120 min, 180 min, 240 min and 300 min to overnight, followed by fixation and permeabilization. Intracellular PCSK9-HT was labeled with 5 µM of TMR ligand for 15 min. The cells were fixed and permeabilized at various time points, from 30 min, 60 min, 120 min, 180 min, and 240 min to 300 min. The cells were counter-stained with LDLR and subcellular organelle markers. Co-localization of internalized or intracellular PCSK9-HT with LDLR and subcellular organelles was visualized by confocal fluorescence imaging.

Fig. (3) Subcellular localization of intracellular PCSK9. 14-13-E4 cells were labeled with 5 µM TMR (Red) for 15 min. The unbound ligand was washed off. Cells were fixed (4% Para-formaldehyde, 0.2 M Sucrose in PBS) and permeabilized (0.1% Triton X-100 in PBS) at various time points as indicated. The cells were then counterstained with different subcellular organelle markers (Green), including PDI (ER), Golgin97 (Golgi complex), EEA1 (early endosome), and LAMP1 (lysosome). The images were acquired with an Evotech Opera Confocal High Content Imager with appropriate fluorescence filters. Co-localization of PCSK9 with each organelle is indicated by arrows. Intracellular PCSK9 is primarily located in the ER and Golgi at the time of labeling, and eventually moves to lysosomes. Co-localization of intracellular PCSK9 with early endosomes is observed as well.

A different strategy of labeling was used to track the internalized PCSK9 pool (Fig. 2). The 14-13-E4 cells were labeled with Alexa Fluor 488 for various time points, ranging from 30 to 300 min and including overnight incubation, followed by fixation and permeabilization. The same set of markers for subcellular organelles as employed for the intracellular PCSK9 pool was applied to locate the Alexa Fluor 488 labeled internalized PCSK9. The localization of the internalized PCSK9 pool was distinct from that of the intracellular PCSK9 (Fig. 4). Alexa Fluor 488 labeled PCSK9 appeared on the cell surface shortly after labeling (Fig. 4 and Supplement movie 1). Trafficking of the internalized PCSK9 from the cell membrane to the endosome/lysosome was observed after 300 min to overnight labeling of secreted PCSK9. The lack of localization of the internalized PCSK9 in the ER or Golgi further confirmed that the labeling was solely on secreted PCSK9.

Fig. (4) Subcellular localization of internalized PCSK9. 14-13-E4 cells were labeled with 0.2 µM Alexa Fluor 488 (Green) for various times (as indicated) followed by fixation (4% para-formaldehyde, 0.2 M Sucrose in PBS) and permeabilization (0.1% Triton X-100 in PBS). The cells were then counterstained for different subcellular organelles (Red), including PDI (ER), Golgin97 (Golgi complex), EEA1 (early endosome), and LAMP1 (lysosome). The images were acquired with an Evotech Opera Confocal High Content Imager with appropriate fluorescence filters. Co-localization of PCSK9 with each organelle is indicated by arrows. Trafficking of internalized PCSK9 from the cell membrane to endosomes-lysosomes was observed.

PCSK9 has become an appealing target for the treatment of dyslipidemia due to the findings that it binds the LDL receptor (LDLR) thus affecting LDL uptake [11]. Secreted PCSK9 can bind the LDLR on the cell surface. The PCSK9-LDLR complex is internalized into endosome and degraded in the lysosome. It has also been claimed that newly synthesized PCSK9 may bind LDLR prior to secretion, therefore leading to direct LDLR degradation in the lysosome [19Maxwell KN, Fisher EA, Breslow JL. Overexpression of PCSK9 accelerates the degradation of the LDLR in a post-endoplasmic reticulum compartment Proc Natl Acad Sci USA 2005; 102: 2069-74.-21Poirier S, Mayer G, Poupon V, et al. Dissection of the endogenous cellular pathways of PCSK9-induced low density lipoprotein receptor degradation evidence for an intracellular route J Biol Chem 2009; 284: 28856-64.]. However, this proposed "PCSK9 intracellular pathway" was established with an over-expression system and was not validated in an in vivo setting. To shed light on PCSK9 trafficking through these two pathways, we examined co-localization of LDLR with these two pools of PCSK9 using HaloTag technology. The experiments were carried out as described above (Fig. 2). Cells with a TMR labeled intracellular pool of PCSK9 or an Alexa Fluor 488 labeled internalized pool of PCSK9 were counterstained with antibodies against LDLR as well as different markers for sub-cellular organelles at various time points. Consistent with the reported results, the secreted PCSK9 co-localized with LDLR in the early endosome and lysosome (Fig. 5A). Co-localization of PCSK9 and LDLR appeared in the early endosome at about 120 min of Alexa Fluor 488 labeling, and continued to show endosome co-localization at later time points as well. Meanwhile lysosome co-localization was observed after longer incubation time at 300 min of Alexa Fluor 488 labeling, which agrees with a trafficking pattern of the PCSK9-LDLR complex from early endosomes to lysosomes. For the TMR labeled intracellular pool of PCSK9, co-localization with LDLR was observed in the ER and Golgi at 30 min post-labeling. Similar to Alexa Fluor 488 labeling, co-localization of PCSK9 and LDLR in the early endosome and lysosome was also observed at a later time point at about 300 min post-labeling (Fig. 5B). In the current experimental design, however, we were not able to differentiate whether the TMR labeled PCSK9 located in early endosome/lysosome had been secreted prior to entering the early endosome or lysosome. Further investigation is needed to address this question.

Fig. (5) Co-localization of PCSK9 with LDLR. A. 14-13-E4 cells were labeled with 0.2 µM Alexa Fluor 488 (Green) as in Fig. (4). B. 14-13-E4 cells were labeled with 5 mM TMR (Red) as in Fig. (3). Following fixation and permeabilization, cells were counter-stained for LDLR and subcellular organelle markers (red in A, green in B). The images were acquired with an Evotech Opera Confocal High Content Imager with appropriate fluorescence filters. Intracellular PCSK9 and LDLR co-localized in the ER and Golgi complex at early time points, and co-localized in lysosomes at later time points. Secreted/internalized PCSK9 mostly co-localized with LDLR in endosomes and lysosomes.