An Unbiased Mass Spectrometry Approach Identifies Glypican-3 as an Interactor of Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) and Low Density Lipoprotein Receptor (LDLR) in Hepatocellular Carcinoma Cells

Abstract

The mechanism of LDL receptor (LDLR) degradation mediated by the proprotein convertase subtilisin/kexin type 9 (PCSK9) has been extensively studied; however, many steps within this process remain unclear and still require characterization. Recent studies have shown that PCSK9 lacking its Cys/His-rich domain can still promote LDLR internalization, but the complex does not reach the lysosome suggesting the presence of an additional interaction partner(s). In this study we carried out an unbiased screening approach to identify PCSK9-interacting proteins in the HepG2 cells' secretome using co-immunoprecipitation combined with mass spectrometry analyses. Several interacting proteins were identified, including glypican-3 (GPC3), phospholipid transfer protein, matrilin-3, tissue factor pathway inhibitor, fibrinogen-like 1, and plasminogen activator inhibitor-1. We then validated these interactions by co-immunoprecipitation and Western blotting. Furthermore, functional validation was examined by silencing each candidate protein in HepG2 cells using short hairpin RNAs to determine their effect on LDL uptake and LDLR levels. Only GPC3 and phospholipid transfer protein silencing in HepG2 cells significantly increased LDL uptake in these cells and displayed higher total LDLR protein levels compared with control cells. Moreover, our study provides the first evidence that GPC3 can modulate the PCSK9 extracellular activity as a competitive binding partner to the LDLR in HepG2 cells.

Keywords: HepG2 cells; LDL receptor (LDLR); glypican-3; knockdown; lentivirus; low density lipoprotein (LDL); mass spectrometry (MS); proprotein convertase subtilisin/kexin type 9 (PCSK9); secretome; short hairpin RNA (shRNA).

FIGURE 1.

Stable overexpression of hPCSK9-V5 in HepG2 cells. A, PCSK9 mRNA expression levels from QPCR analysis on RNA extracts from HepG2 cells stably expressing an empty construct pIR and pIR/hPCSK9-V5. Results are the average of four independent experiments relative to HepG2 control pIR with the standard deviation as error bars. Results are normalized using human actin housekeeping gene. *, p < 0.05. B, Western blot (WB) of total cell lysate (25 μg of protein) and conditioned cell media from stable HepG2 empty pIR and pIR/hPCSK9-V5 cells were resolved on SDS-PAGE, blotted onto a nitrocellulose membrane, and incubated with primary mouse anti-V5.

FIGURE 2.

PCSK9 immunoprecipitation from HepG2 cell media. Concentrated conditioned media from HepG2 empty pIR and pIR/hPCSK9-V5 cells (100 μg of protein) were incubated and precipitated overnight with protein G magnetic beads cross-linked with either mouse normal IgG or mouse anti-V5. Immunoprecipitates were washed and separated on SDS-PAGE and probed by Western blotting (WB) with anti-V5. Input lane corresponds to 10% of the total amount of proteins used for each immunoprecipitation (IP).

FIGURE 3.

Mass spectrometry analysis of immunoprecipitated hPCSK9-V5 from HepG2 cells' secretome. List of secreted proteins identified by MS analysis from HepG2 cell secretome stably overexpressing V5-tagged hPCSK9. Unused ProtScore (protein confidence) across all samples was used to calculate the enrichment fold ratio from 1- to 10-fold (ranked as PCSK9-IP anti-V5/PCSK9-IP normal anti-IgG).

FIGURE 4.

Co-immunoprecipitation of PCSK9 with six selected binding partners. A, PCSK9-V5 was immunoprecipitated (IP) as described in Fig. 2 and analyzed by Western blotting (WB) with anti-V5 as a positive control, anti-GPC3, anti-PLTP, anti-MATN3, anti-TFPI, anti-FGL1, and anti-PAI-1. B, GPC3 and PLTP were immunoprecipitated from HepG2 pIR/hPCSK9-V5 cell-conditioned media (100 μg of protein) using anti-GPC3 mouse monoclonal antibody or anti-PLTP, and the presence of PCSK9 in the immunoprecipitates was assessed with a V5 antibody. C, GPC3 was immunoprecipitated from conditioned media of HepG2 cells overexpressing hPCSK9 (no V5 tag), analyzed, and probed by Western blotting for PCSK9 using rabbit anti-PCSK9. Input lane corresponds to 10% of the total amount of conditioned media used for each immunoprecipitation. Results are representative of three independent experiments.

FIGURE 5.

Extracellular GPC3 interaction with PCSK9ΔCHRD. Conditioned media from HepG2 cells overexpressing hPCSK9ΔCHRD-V5 (1st to 3rd lanes) or CHRD-V5 (4th to 6th lanes) were collected to perform GPC3 immunoprecipitation (IP) as described in Figs. 2 and 4. Immunoprecipitates were resolved on SDS-PAGE and probed by Western blotting (WB) with a V5 antibody. Results are representative of three independent experiments.

FIGURE 6.

Co-immunoprecipitation of intracellular GPC3 with PCSK9 and LDLR in Huh7 cells. Western blotting (WB) of cell lysates (25 μg) from Huh7 cells overexpressing PCSK9 WT-V5, LDLR-V5, or GPC3-HA. Intracellular protein expression levels from each co-expression conditions were confirmed from 1st to 8th lanes using an anti-V5 (A) and anti-HA (B). GPC3 was immunoprecipitated (IP: anti-HA), and the co-immunoprecipitation of PCSK9 and LDLR was confirmed using an anti-V5 (C, 5th, 7th, and 8th lanes). GPC3 protein levels from the pulldown was analyzed by Western blotting using an anti-HA in each cell lysate (D). Cell lysate β-actin levels were used for normalization. Results are representative of three independent experiments.

FIGURE 7.

mRNA expression levels from stable knockdown HepG2 cells. QPCR analysis on RNA extracts from HepG2 cells expressing a nonspecific shRNA control (shNon-target) or an shRNA targeting GPC3, PLTP, MATN3, FGL1, PAI1, and TFPI. Results are the average of two independent experiments relative to HepG2 control shNon-target with the standard deviation as error bars. Results are normalized using the human actin housekeeping gene.

FIGURE 8.

Differences in basal DiI-LDL uptake in stable knockdown HepG2 cells. Each stable knockdown cell line was grown for 24 h followed by serum deprivation for 24 h and incubated with DiI-LDL for 4 h. Raw fluorescence units were normalized by cell numbers per well using CyQUANT® fluorescence units in relation to control shNon-target HepG2 cells. Results are the average of three independent experiments with the standard deviation as error bars. *, p < 0.05; **, p < 0.01.

FIGURE 9.

LDLR mRNA expression and total protein levels in stable GPC3 and PLTP knockdown HepG2 cells. A, QPCR analysis of LDLR mRNA in HepG2 shGPC3 and shPLTP stable cell lines relative to shNT cells. Results are shown as three independent experiments with the standard deviation as error bars. Results are normalized using human actin housekeeping gene (*, p < 0.05). B, total cell lysates (25 μg) from stable HepG2 shNT, shGPC3, and shPLTP cells were resolved on SDS-PAGE and subjected to Western blotting using primary goat anti-LDLR and anti-β-actin. Duplicates for each condition are shown, and results are representative of three independent experiments. Protein levels relative to shNT cells were normalized on β-actin levels.

FIGURE 10.

DiI-LDL uptake response curve to hPCSK9 RS in absence of GPC3 and PLTP. Purified human recombinant PCSK9 RS was added to HepG2 cell media at a final concentration of 0.001–20 μg/ml (hPCSK9 RS) followed by the addition of DiI-LDL as described in Fig. 8. DiI-LDL uptake in HepG2 cells shNT versus shGPC3 (A) and shPLTP (B) in the presence of hPCSK9-RS. DiI-LDL incorporation is normalized with CyQUANT® raw fluorescence units (RFU) relative to maximal DiI-LDL incorporation observed in the absence of recombinant hPCSK9 RS. Results are the average of three independent experiments with the standard error of the mean as error bars.

FIGURE 11.

Effective concentration of hPCSK9 RS. Concentrations of hPCSK9 RS necessary to reduce DiI-LDL uptake by 50% in control shNT, shGPC3, and shPLTP are shown in μg/ml. Results are the average of three independent experiments with the standard error of the mean as error bars. *, p < 0.05, and n.s. means non-significant.

FIGURE 12.

GPC3 and PLTP knockdown impact on extracellular hPCSK9 RS activity in HepG2 cells. Total cell lysates (25 μg) from HepG2 shNT and shGPC3 (A) and shPLTP (B) incubated for 6 h with 10 μg/ml hPCSK9 RS (RS) were resolved on SDS-PAGE and analyzed by Western blotting with anti-LDLR and anti-β-actin. Total LDLR protein levels were quantitated, normalized based on β-actin levels, and shown as histograms below a membrane representative of the experiment. LDLR protein levels in each cell line incubated with hPCSK9 RS (RS) are relative to its respective control (Cnt). Results are the average with the standard deviation as error bars from three independent experiments (A) (*, p < 0.05) and two independent experiments (B) (n.s. means non-significant).

FIGURE 13.

GPC3 impact on extracellular PCSK9 activity in Huh7 cells. Western blotting of total cell lysates (25 μg) from Huh7 cells overexpressing LDLR, GPC3, or both. Huh7 cells were incubated in the presence or in absence of hPCSK9 WT-V5-conditioned media for 6 h. LDLR and GPC3 protein levels were probed, and cell lysate β-actin levels were used for normalization. Quantification is representative of two independent experiments.

FIGURE 14.

In situ hybridization evidence for developmentally regulated GPC3 expression in the mouse. A and B, mouse embryos at mid-gestation stage. X-ray film autoradiography showing mRNA labeling seen as bright under dark field illumination. High level of GPC3 expression is evident in the mandible mesenchyme. Down-regulated GPC3 expression is evident in the liver from high levels in e10 to moderate in e15 embryos. C and D, postnatal GPC3 expression in the kidney and intestine, up-regulated from low levels in p1 to moderate and high in p10; increasing levels of GPC3 expression in the membranes of the pleura, pericardium, and periosteum. Moderate level of GPC3 expression is noted in the liver of a newborn mouse (in C) and low level in postnatal p10 (in D). E, undetectable GPC3 expression in the adult mouse liver. Abbreviations used are as follows: H, heart; He, head; K, kidney; In, intestine; Li, liver; MPc, membranes of the pericardium; MPe, membranes of the periosteum; MPl, membranes of the pleura; Mo, molar with the membranes. Magnifications: A and B, ×8; C, ×4; D and E, ×2.

FIGURE 15.

PCSK9 and GPC3 interaction model. Illustration of PCSK9 extracellular activity on LDLR degradation in the absence (A) or presence (B) of intracellular and extracellular GPC3 in HepG2 and Huh7 cells. A, cell-surface LDLR interaction with PCSK9 leads to its internalization and its degradation via the endosomal/lysosomal pathway. This action results in lower LDLR at the cell surface with an accumulation of circulating LDL particles. B, early interaction of pro-PCSK9-GPC3 in the ER is illustrated based on co-immunoprecipitation results presented in Fig. 6. The impact of this intracellular interaction on pro-PCSK9 remains to be determined. We also propose that the presence of either cell membrane or secreted/soluble GPC3 can interact with PCSK9 and prevent its binding to cell-surface LDLR. This competitive binding of GPC3 and PCSK9 to the LDLR reduces PCSK9 extracellular activity resulting in higher LDLR levels and higher LDL uptake.