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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*

  • Author Footnotes
    1 Recipient of studentship support from the Fonds de Recherche du Québec-Santé (FRQS).
    Kévin Ly
    Footnotes
    1 Recipient of studentship support from the Fonds de Recherche du Québec-Santé (FRQS).
    Affiliations
    From the Institut de Pharmacologie de Sherbrooke, Department of Surgery/Urology Division, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Quebec J1H5N4 and
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  • Rachid Essalmani
    Affiliations
    the Institut de Recherches Cliniques de Montréal, Affiliated with Université de Montréal, Montréal, Quebec H2W 1R7, Canada
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  • Roxane Desjardins
    Affiliations
    From the Institut de Pharmacologie de Sherbrooke, Department of Surgery/Urology Division, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Quebec J1H5N4 and
    Search for articles by this author
  • Nabil G. Seidah
    Affiliations
    the Institut de Recherches Cliniques de Montréal, Affiliated with Université de Montréal, Montréal, Quebec H2W 1R7, Canada
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  • Robert Day
    Correspondence
    To whom correspondence should be addressed: Institut de Pharmacologie de Sherbrooke (IPS), Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, 3001 12e Ave. Nord, Sherbrooke, Québec J1H5N4, Canada. Tel.: 819-821-8000 (Ext. 75428); Fax: 819-820-6886; E-mail: .
    Affiliations
    From the Institut de Pharmacologie de Sherbrooke, Department of Surgery/Urology Division, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Quebec J1H5N4 and
    Search for articles by this author
  • Author Footnotes
    * This work was supported in part by Fondation Leducq Grant 13 CVD 03 (to R. D. and N. G. S.), by Canada Research Chair 126684 (to N. G. S.), and in part by a Pfizer ASPIRE CV IIR Grant (to N. G. S.). The authors declare that they have no conflicts of interest with the contents of this article.
    1 Recipient of studentship support from the Fonds de Recherche du Québec-Santé (FRQS).
Open AccessPublished:October 07, 2016DOI:https://doi.org/10.1074/jbc.M116.746883

      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.
      Hypercholesterolemia, characterized by elevated plasma levels of low density lipoprotein (LDL)-cholesterol (LDL-C),
      The abbreviations used are: LDL-C, low density lipoprotein-cholesterol; CHRD, Cys/His-rich domain; GPC3, glypican-3; PLTP, phospholipid transfer protein; LDLR, LDL receptor; FH, familial hypercholesterolemia; ER, endoplasmic reticulum; AnxA2, annexin A2; co-IP, co-immunoprecipitation; TFPI, tissue factor pathway inhibitor; UHPLC-MS/MS, micro-flow high performance liquid chromatography coupled with tandem mass spectrometry; shNT, shRNA non-target; DiI-LDL, 1,1′-dioctadecyl-3,3,3′-3′-tetramethylindocarbocyanine perchlorate-labeled LDL; shGPC3, stable GPC3 knockdown; shPLTP, stable PLTP knockdown; hPCSK9 RS, hPCSK9 R218S; IP, immunoprecipitation; QPCR, quantitative PCR; HCC, human hepatocellular carcinoma.
      3The abbreviations used are: LDL-C, low density lipoprotein-cholesterol; CHRD, Cys/His-rich domain; GPC3, glypican-3; PLTP, phospholipid transfer protein; LDLR, LDL receptor; FH, familial hypercholesterolemia; ER, endoplasmic reticulum; AnxA2, annexin A2; co-IP, co-immunoprecipitation; TFPI, tissue factor pathway inhibitor; UHPLC-MS/MS, micro-flow high performance liquid chromatography coupled with tandem mass spectrometry; shNT, shRNA non-target; DiI-LDL, 1,1′-dioctadecyl-3,3,3′-3′-tetramethylindocarbocyanine perchlorate-labeled LDL; shGPC3, stable GPC3 knockdown; shPLTP, stable PLTP knockdown; hPCSK9 RS, hPCSK9 R218S; IP, immunoprecipitation; QPCR, quantitative PCR; HCC, human hepatocellular carcinoma.
      is the major risk factor for atherosclerosis and cardiovascular diseases. LDL particles, which carry most of body cholesterol, are eliminated from the circulation mainly via their binding to the liver hepatocyte cell-surface LDL receptors (LDLR). This interaction occurs between the apolipoprotein B-100 (apoB-100) on LDL particles and the LDLR. Mutations in both LDLR or ApoB genes are associated with familial hypercholesterolemia (FH), an autosomal dominant genetic disorder (
      • Abifadel M.
      • Varret M.
      • Rabès J.-P.
      • Allard D.
      • Ouguerram K.
      • Devillers M.
      • Cruaud C.
      • Benjannet S.
      • Wickham L.
      • Erlich D.
      • Derré A.
      • Villéger L.
      • Farnier M.
      • Beucler I.
      • Bruckert E.
      • et al.
      Mutations in PCSK9 cause autosomal dominant hypercholesterolemia.
      ). In 2003, the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene (
      • Seidah N.G.
      • Benjannet S.
      • Wickham L.
      • Marcinkiewicz J.
      • Jasmin S.B.
      • Stifani S.
      • Basak A.
      • Prat A.
      • Chretien M.
      The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): liver regeneration and neuronal differentiation.
      ) was the third locus validated to be associated with FH (
      • Abifadel M.
      • Varret M.
      • Rabès J.-P.
      • Allard D.
      • Ouguerram K.
      • Devillers M.
      • Cruaud C.
      • Benjannet S.
      • Wickham L.
      • Erlich D.
      • Derré A.
      • Villéger L.
      • Farnier M.
      • Beucler I.
      • Bruckert E.
      • et al.
      Mutations in PCSK9 cause autosomal dominant hypercholesterolemia.
      ) and the last member of the proprotein convertase family identified (
      • Seidah N.G.
      • Benjannet S.
      • Wickham L.
      • Marcinkiewicz J.
      • Jasmin S.B.
      • Stifani S.
      • Basak A.
      • Prat A.
      • Chretien M.
      The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): liver regeneration and neuronal differentiation.
      ).
      PCSK9 is mostly expressed and secreted into the plasma from the liver and is also found at lower levels in small intestine and kidneys (
      • Seidah N.G.
      • Benjannet S.
      • Wickham L.
      • Marcinkiewicz J.
      • Jasmin S.B.
      • Stifani S.
      • Basak A.
      • Prat A.
      • Chretien M.
      The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): liver regeneration and neuronal differentiation.
      ,
      • Zaid A.
      • Roubtsova A.
      • Essalmani R.
      • Marcinkiewicz J.
      • Chamberland A.
      • Hamelin J.
      • Tremblay M.
      • Jacques H.
      • Jin W.
      • Davignon J.
      • Seidah N.G.
      • Prat A.
      Proprotein convertase subtilisin/kexin type 9 (PCSK9): hepatocyte-specific low density lipoprotein receptor degradation and critical role in mouse liver regeneration.
      ). It is synthesized in the endoplasmic reticulum (ER) as a zymogen pro-PCSK9 (75 kDa) where it must undergo an autocatalytic cleavage of its prodomain (13 kDa) to exit this compartment and be secreted in its mature form (62 kDa). Mature PCSK9 remains non-covalently associated with its cleaved prodomain (
      • Seidah N.G.
      • Benjannet S.
      • Wickham L.
      • Marcinkiewicz J.
      • Jasmin S.B.
      • Stifani S.
      • Basak A.
      • Prat A.
      • Chretien M.
      The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): liver regeneration and neuronal differentiation.
      ,
      • Benjannet S.
      • Rhainds D.
      • Essalmani R.
      • Mayne J.
      • Wickham L.
      • Jin W.
      • Asselin M.-C.
      • Hamelin J.
      • Varret M.
      • Allard D.
      • Trillard M.
      • Abifadel M.
      • Tebon A.
      • Attie A.D.
      • Rader D.J.
      • et al.
      NARC-1/PCSK9 and its natural mutants: zymogen cleavage and effects on the low density lipoprotein (LDL) receptor and LDL cholesterol.
      ), and this tight complex is secreted as a catalytically inactive serine protease (
      • Cunningham D.
      • Danley D.E.
      • Geoghegan K.F.
      • Griffor M.C.
      • Hawkins J.L.
      • Subashi T.A.
      • Varghese A.H.
      • Ammirati M.J.
      • Culp J.S.
      • Hoth L.R.
      • Mansour M.N.
      • McGrath K.M.
      • Seddon A.P.
      • Shenolikar S.
      • Stutzman-Engwall K.J.
      • et al.
      Structural and biophysical studies of PCSK9 and its mutants linked to familial hypercholesterolemia.
      ). The role of PCSK9 in cholesterol metabolism derived from its ability to bind the EGF-A domain of the LDLR (
      • Kwon H.J.
      • Lagace T.A.
      • McNutt M.C.
      • Horton J.D.
      • Deisenhofer J.
      Molecular basis for LDL receptor recognition by PCSK9.
      ) either intracellularly or extracellularly (
      • Poirier S.
      • Mayer G.
      • Poupon V.
      • McPherson P.S.
      • Desjardins R.
      • Ly K.
      • Asselin M.-C.
      • Day R.
      • Duclos F.J.
      • Witmer M.
      • Parker R.
      • Prat A.
      • Seidah N.G.
      Dissection of the endogenous cellular pathways of PCSK9-induced low density lipoprotein receptor degradation: evidence for an intracellular route.
      ), and the PCSK9-LDLR complex is then sorted to the endosome/lysosome pathway for degradation (
      • Nassoury N.
      • Blasiole D.A.
      • Tebon Oler A.
      • Benjannet S.
      • Hamelin J.
      • Poupon V.
      • McPherson P.S.
      • Attie A.D.
      • Prat A.
      • Seidah N.G.
      The cellular trafficking of the secretory proprotein convertase PCSK9 and its dependence on the LDLR.
      ). This action reduces LDLR protein levels at the cell surface and thus decreases the elimination of circulating LDL-C. Therefore, PCSK9 promotes LDLR degradation, and PCSK9 inactivation in mice has shown beneficial effects in lowering total cholesterol levels by as much as 42% in the PCSK9 knock-out mouse model (
      • Zaid A.
      • Roubtsova A.
      • Essalmani R.
      • Marcinkiewicz J.
      • Chamberland A.
      • Hamelin J.
      • Tremblay M.
      • Jacques H.
      • Jin W.
      • Davignon J.
      • Seidah N.G.
      • Prat A.
      Proprotein convertase subtilisin/kexin type 9 (PCSK9): hepatocyte-specific low density lipoprotein receptor degradation and critical role in mouse liver regeneration.
      ,
      • Rashid S.
      • Curtis D.E.
      • Garuti R.
      • Anderson N.N.
      • Bashmakov Y.
      • Ho Y.K.
      • Hammer R.E.
      • Moon Y.A.
      • Horton J.D.
      Decreased plasma cholesterol and hypersensitivity to statins in mice lacking Pcsk9.
      ) or up to 27% in liver-specific PCSK9 knock-out mice (
      • Zaid A.
      • Roubtsova A.
      • Essalmani R.
      • Marcinkiewicz J.
      • Chamberland A.
      • Hamelin J.
      • Tremblay M.
      • Jacques H.
      • Jin W.
      • Davignon J.
      • Seidah N.G.
      • Prat A.
      Proprotein convertase subtilisin/kexin type 9 (PCSK9): hepatocyte-specific low density lipoprotein receptor degradation and critical role in mouse liver regeneration.
      ). Inactivating monoclonal antibodies are now prescribed in lipid clinics worldwide as it was shown that reduction of plasma PCSK9 levels result in ∼60% lowering of plasma LDL-C (
      • Seidah N.G.
      • Awan Z.
      • Chrétien M.
      • Mbikay M.
      PCSK9: a key modulator of cardiovascular health.
      ).
      The cause and effect of PCSK9 on LDL-C levels has been well described, but the underlying mechanism(s) by which PCSK9 directs cell-surface LDLR to late endosome/lysosome degradation lacks details. Previous studies demonstrated that the deletion (
      • Strøm T.B.
      • Holla Ø.L.
      • Tveten K.
      • Cameron J.J.
      • Berge K.E.
      • Leren T.P.
      Disrupted recycling of the low density lipoprotein receptor by PCSK9 is not mediated by residues of the cytoplasmic domain.
      ) and/or substitution (
      • Holla Ø.L.
      • Strøm T.B.
      • Cameron J.
      • Berge K.E.
      • Leren T.P.
      A chimeric LDL receptor containing the cytoplasmic domain of the transferrin receptor is degraded by PCSK9.
      ,
      • Canuel M.
      • Sun X.
      • Asselin M.-C.
      • Paramithiotis E.
      • Prat A.
      • Seidah N.G.
      Proprotein convertase subtilisin/kexin type 9 (PCSK9) can mediate degradation of the low density lipoprotein receptor-related protein 1 (LRP-1).
      ) of LDLR's cytosolic tail does not prevent PCSK9's ability to mediate LDLR degradation. Moreover, the autosomal recessive hypercholesterolemia adaptor protein, responsible for LDLR internalization through the clathrin-dependent mechanism, is also not absolutely necessary for LDLR degradation mediated by PCSK9 (
      • Norman D.
      • Sun X.M.
      • Bourbon M.
      • Knight B.L.
      • Naoumova R.P.
      • Soutar A.K.
      Characterization of a novel cellular defect in patients with phenotypic homozygous familial hypercholesterolemia.
      ). It was also hypothesized that the membrane-bound PCSK9-LDLR complex is first shed into a soluble form upon entry into acidic endosomes via a cathepsin-like cysteine protease inhibited by E64, which may facilitate its efficacious degradation in lysosomes (
      • Leren T.P.
      Sorting an LDL receptor with bound PCSK9 to intracellular degradation.
      ). Taken together, we can infer the presence of additional membrane-bound or secreted cellular proteins involved in this mechanism or the presence of another internalization pathway requiring binding partners that remain unidentified.
      Additionally, deletion of PCSK9's Cys/His-rich domain (CHRD) does not affect its capacity to bind and internalize LDLR, but its presence is required to direct the complex to lysosomes and disrupt its recycling (
      • Cunningham D.
      • Danley D.E.
      • Geoghegan K.F.
      • Griffor M.C.
      • Hawkins J.L.
      • Subashi T.A.
      • Varghese A.H.
      • Ammirati M.J.
      • Culp J.S.
      • Hoth L.R.
      • Mansour M.N.
      • McGrath K.M.
      • Seddon A.P.
      • Shenolikar S.
      • Stutzman-Engwall K.J.
      • et al.
      Structural and biophysical studies of PCSK9 and its mutants linked to familial hypercholesterolemia.
      ,
      • Zhang D.-W.
      • Garuti R.
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      • Hobbs H.H.
      Structural requirements for PCSK9-mediated degradation of the low density lipoprotein receptor.
      • Ni Y.G.
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      • Pandit S.
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      • Ruggeri L.
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      • Ehrhardt A.
      • Lewis D.
      • Fisher T.S.
      • Ha S.
      • et al.
      A proprotein convertase subtilisin-like/kexin type 9 (PCSK9) C-terminal domain antibody antigen-binding fragment inhibits PCSK9 internalization and restores low density lipoprotein uptake.
      ,
      • Holla Ø.L.
      • Cameron J.
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      • Strøm T.B.
      • Berge K.E.
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      • Leren T.P.
      Role of the C-terminal domain of PCSK9 in degradation of the LDL receptors.
      ,
      • Saavedra Y.G.
      • Day R.
      • Seidah N.G.
      The M2 module of the Cys-His-rich domain (CHRD) of PCSK9 protein is needed for the extracellular low density lipoprotein receptor (LDLR) degradation pathway.
      • Weider E.
      • Susan-Resiga D.
      • Essalmani R.
      • Hamelin J.
      • Asselin M.-C.
      • Ashraf Y.
      • Wycoff K.L.
      • Zhang J.
      • Prat A.
      • Seidah N.G.
      Proprotein convertase subtilisin/Kexin type 9 (PCSK9) single domain antibodies are potent inhibitors of LDL receptor degradation.
      ). It was proposed that in mildly acidic conditions, the positively charged His-rich PCSK9 CHRD is required to keep the PCSK9 bound to the LDLR ligand-binding domain to reroute the complex to lysosomes (
      • Tveten K.
      • Holla Ø.L.
      • Cameron J.
      • Strøm T.B.
      • Berge K.E.
      • Laerdahl J.K.
      • Leren T.P.
      Interaction between the ligand-binding domain of the LDL receptor and the C-terminal domain of PCSK9 is required for PCSK9 to remain bound to the LDL receptor during endosomal acidification.
      ). However, it is also possible that other proteins could interact with the PCSK9 CHRD and act as a co-receptor for LDLR trafficking. Several studies have demonstrated that such interaction occurs extracellularly with the extrahepatic protein annexin A2 (AnxA2) acting as an endogenous inhibitor of PCSK9 via its ability to bind the CHRD and thus prevent its interaction with the LDLR (
      • Mayer G.
      • Poirier S.
      • Seidah N.G.
      Annexin A2 is a C-terminal PCSK9-binding protein that regulates endogenous low density lipoprotein receptor levels.
      ,
      • Seidah N.G.
      • Poirier S.
      • Denis M.
      • Parker R.
      • Miao B.
      • Mapelli C.
      • Prat A.
      • Wassef H.
      • Davignon J.
      • Hajjar K.A.
      • Mayer G.
      Annexin A2 is a natural extrahepatic inhibitor of the PCSK9-induced LDL receptor degradation.
      • Ly K.
      • Saavedra Y.G.
      • Canuel M.
      • Routhier S.
      • Desjardins R.
      • Hamelin J.
      • Mayne J.
      • Lazure C.
      • Seidah N.G.
      • Day R.
      Annexin A2 reduces PCSK9 protein levels via a translational mechanism and interacts with the M1 and M2 domains of PCSK9.
      ).
      Although many studies have focused on the identification of new proteins involved in the PCSK9-LDLR degradation pathway, such as AnxA2, LRP1, APLP2, apoB-100, sec24a, GRP94, etc. (
      • Canuel M.
      • Sun X.
      • Asselin M.-C.
      • Paramithiotis E.
      • Prat A.
      • Seidah N.G.
      Proprotein convertase subtilisin/kexin type 9 (PCSK9) can mediate degradation of the low density lipoprotein receptor-related protein 1 (LRP-1).
      ,
      • Mayer G.
      • Poirier S.
      • Seidah N.G.
      Annexin A2 is a C-terminal PCSK9-binding protein that regulates endogenous low density lipoprotein receptor levels.
      • Seidah N.G.
      • Poirier S.
      • Denis M.
      • Parker R.
      • Miao B.
      • Mapelli C.
      • Prat A.
      • Wassef H.
      • Davignon J.
      • Hajjar K.A.
      • Mayer G.
      Annexin A2 is a natural extrahepatic inhibitor of the PCSK9-induced LDL receptor degradation.
      ,
      • Ly K.
      • Saavedra Y.G.
      • Canuel M.
      • Routhier S.
      • Desjardins R.
      • Hamelin J.
      • Mayne J.
      • Lazure C.
      • Seidah N.G.
      • Day R.
      Annexin A2 reduces PCSK9 protein levels via a translational mechanism and interacts with the M1 and M2 domains of PCSK9.
      ,
      • Sun H.
      • Samarghandi A.
      • Zhang N.
      • Yao Z.
      • Xiong M.
      • Teng B.-B.
      Proprotein convertase subtilisin/kexin type 9 interacts with apolipoprotein B and prevents its intracellular degradation, irrespective of the low density lipoprotein receptor.
      ,
      • Poirier S.
      • Mamarbachi M.
      • Chen W.-T.
      • Lee A.S.
      • Mayer G.
      GRP94 regulates circulating cholesterol levels through blockade of PCSK9-induced LDLR degradation.
      • Butkinaree C.
      • Canuel M.
      • Essalmani R.
      • Poirier S.
      • Benjannet S.
      • Asselin M.-C.
      • Roubtsova A.
      • Hamelin J.
      • Marcinkiewicz J.
      • Chamberland A.
      • Guillemot J.
      • Mayer G.
      • Sisodia S.S.
      • Jacob Y.
      • Prat A.
      • Seidah N.G.
      Amyloid precursor-like protein 2 and sortilin do not regulate the PCSK9 convertase-mediated low density lipoprotein receptor degradation but interact with each other.
      ), several questions need to be addressed, including the mechanism leading the cell-surface PCSK9-LDLR complex to lysosomal degradation. Herein, we sought to identify novel extracellular interaction partners that could participate and regulate the PCSK9-LDLR complex formation and hence LDLR degradation.
      To identify these binding partners we performed co-immunoprecipitation (co-IP) of PCSK9 combined with a mass spectrometry (MS) analysis on the HepG2 cells' secretome. Our studies identified the novel intracellular and extracellular GPC3 interaction with PCSK9 in human hepatocellular carcinoma (HCC) cell lines HepG2 and Huh7. More importantly, we demonstrated that this interaction reduces the PCSK9 extracellular activity on LDLR degradation.

      Discussion

      There is a good case to be made for the presence of additional interaction partners involved in the regulation of LDLR degradation by PCSK9 (
      • Cunningham D.
      • Danley D.E.
      • Geoghegan K.F.
      • Griffor M.C.
      • Hawkins J.L.
      • Subashi T.A.
      • Varghese A.H.
      • Ammirati M.J.
      • Culp J.S.
      • Hoth L.R.
      • Mansour M.N.
      • McGrath K.M.
      • Seddon A.P.
      • Shenolikar S.
      • Stutzman-Engwall K.J.
      • et al.
      Structural and biophysical studies of PCSK9 and its mutants linked to familial hypercholesterolemia.
      ,
      • Seidah N.G.
      • Awan Z.
      • Chrétien M.
      • Mbikay M.
      PCSK9: a key modulator of cardiovascular health.
      ,
      • Strøm T.B.
      • Holla Ø.L.
      • Tveten K.
      • Cameron J.J.
      • Berge K.E.
      • Leren T.P.
      Disrupted recycling of the low density lipoprotein receptor by PCSK9 is not mediated by residues of the cytoplasmic domain.
      • Holla Ø.L.
      • Strøm T.B.
      • Cameron J.
      • Berge K.E.
      • Leren T.P.
      A chimeric LDL receptor containing the cytoplasmic domain of the transferrin receptor is degraded by PCSK9.
      ,
      • Norman D.
      • Sun X.M.
      • Bourbon M.
      • Knight B.L.
      • Naoumova R.P.
      • Soutar A.K.
      Characterization of a novel cellular defect in patients with phenotypic homozygous familial hypercholesterolemia.
      ,
      • Ni Y.G.
      • Condra J.H.
      • Orsatti L.
      • Shen X.
      • Di Marco S.
      • Pandit S.
      • Bottomley M.J.
      • Ruggeri L.
      • Cummings R.T.
      • Cubbon R.M.
      • Santoro J.C.
      • Ehrhardt A.
      • Lewis D.
      • Fisher T.S.
      • Ha S.
      • et al.
      A proprotein convertase subtilisin-like/kexin type 9 (PCSK9) C-terminal domain antibody antigen-binding fragment inhibits PCSK9 internalization and restores low density lipoprotein uptake.
      ,
      • Holla Ø.L.
      • Cameron J.
      • Tveten K.
      • Strøm T.B.
      • Berge K.E.
      • Laerdahl J.K.
      • Leren T.P.
      Role of the C-terminal domain of PCSK9 in degradation of the LDL receptors.
      ). The routing of the PCSK9-LDLR complex from its internalization to its degradation through the lysosomal pathway is still not fully defined. Mass spectrometry analyses have previously been performed to identify novel interaction partners of PCSK9 (
      • Mayer G.
      • Poirier S.
      • Seidah N.G.
      Annexin A2 is a C-terminal PCSK9-binding protein that regulates endogenous low density lipoprotein receptor levels.
      ,
      • Denis N.
      • Palmer-Smith H.
      • Elisma F.
      • Busuttil A.
      • Wright T.G.
      • Bou Khalil M.
      • Prat A.
      • Seidah N.G.
      • Chrétien M.
      • Mayne J.
      • Figeys D.
      Quantitative proteomic analysis of PCSK9 gain of function in human hepatic HuH7 cells.
      ) in different cell lines. Such studies used various mass spectrometry approaches (SILAC-MS, shotgun-MS/MS) to analyze extracts of HEK293 cells (
      • Xu W.
      • Liu L.
      • Hornby D.
      c-IAP1 binds and processes PCSK9 protein: linking the c-IAP1 in a TNF-α pathway to PCSK9-mediated LDLR degradation pathway.
      ), subcellular fractions of Huh7 cells (
      • Denis N.
      • Palmer-Smith H.
      • Elisma F.
      • Busuttil A.
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      • Chrétien M.
      • Mayne J.
      • Figeys D.
      Quantitative proteomic analysis of PCSK9 gain of function in human hepatic HuH7 cells.
      ) stably overexpressing PCSK9, or membrane fractions preparations from mouse livers (
      • Canuel M.
      • Sun X.
      • Asselin M.-C.
      • Paramithiotis E.
      • Prat A.
      • Seidah N.G.
      Proprotein convertase subtilisin/kexin type 9 (PCSK9) can mediate degradation of the low density lipoprotein receptor-related protein 1 (LRP-1).
      ). One study has successfully identified the novel partner AnxA2 by performing immunoprecipitation of PCSK9 in the COS-1 cell lysate (
      • Mayer G.
      • Poirier S.
      • Seidah N.G.
      Annexin A2 is a C-terminal PCSK9-binding protein that regulates endogenous low density lipoprotein receptor levels.
      ). It was recently reported that the LDLR does not bind efficiently PCSK9 in the ER because of the presence of a competitive chaperone like-protein GRP94 that prevents such ER interaction (
      • Poirier S.
      • Mamarbachi M.
      • Chen W.-T.
      • Lee A.S.
      • Mayer G.
      GRP94 regulates circulating cholesterol levels through blockade of PCSK9-induced LDLR degradation.
      ). Aside from the latter study, no other reports appeared on the identification of strong and stable interaction partners of PCSK9 from the secretome of HepG2 cells by mass spectrometry analysis. Therefore, this study has focused on the identification of novel extracellular interaction partners of PCSK9 from the conditioned media of HepG2 cells. We initially identified 42 secreted proteins (Fig. 3) based on mass spectrometry analysis of PCSK9 immunoprecipitates from the secretome of HepG2 cells. Six proteins were selected from database data linking them to the following: (i) cholesterol homeostasis; (ii) interactions with LDLR family members; or (iii) an association with plasma LDL-C. It has been shown that cell-surface GPC3, TFPI, and PAI-1 can be internalized through an LRP1-dependent mechanism (
      • Cortese K.
      • Sahores M.
      • Madsen C.D.
      • Tacchetti C.
      • Blasi F.
      Clathrin and LRP-1-independent constitutive endocytosis and recycling of uPAR.
      • Capurro M.I.
      • Shi W.
      • Filmus J.
      LRP1 mediates Hedgehog-induced endocytosis of the GPC3-Hedgehog complex.
      ,
      • Hamik A.
      • Setiadi H.
      • Bu G.
      • McEver R.P.
      • Morrissey J.H.
      Down-regulation of monocyte tissue factor mediated by tissue factor pathway inhibitor and the low density lipoprotein receptor-related protein.
      ,
      • Llorente-Cortés V.
      • Otero-Viñas M.
      • Camino-López S.
      • Llampayas O.
      • Badimon L.
      Aggregated low density lipoprotein uptake induces membrane tissue factor procoagulant activity and microparticle release in human vascular smooth muscle cells.
      • Strickland D.K.
      • Au D.T.
      • Cunfer P.
      • Muratoglu S.C.
      Low-density lipoprotein receptor-related protein-1: role in the regulation of vascular integrity.
      ). TFPI and PLTP have been positively associated with LDL particles in human plasma (
      • Dashty M.
      • Motazacker M.M.
      • Levels J.
      • de Vries M.
      • Mahmoudi M.
      • Peppelenbosch M.P.
      • Rezaee F.
      Proteome of human plasma very low density lipoprotein and low density lipoprotein exhibits a link with coagulation and lipid metabolism.
      ,
      • Murdoch S.J.
      • Carr M.C.
      • Kennedy H.
      • Brunzell J.D.
      • Albers J.J.
      Selective and independent associations of phospholipid transfer protein and hepatic lipase with the LDL subfraction distribution.
      ), whereas PLTP has been mostly studied for its important role in HDL metabolism. FGL1 knock-out mice exhibit lower free fatty acid levels and total plasma cholesterol levels (
      • Demchev V.
      • Malana G.
      • Vangala D.
      • Stoll J.
      • Desai A.
      • Kang H.W.
      • Li Y.
      • Nayeb-Hashemi H.
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      • Cohen D.E.
      • Ukomadu C.
      Targeted deletion of fibrinogen like protein 1 reveals a novel role in energy substrate utilization.
      ), and MATN family exhibit epidermal growth factor-like domain, which could possibly interact with PCSK9 such as the one stipulated in an MATN2 EGF-like domain patent (United States patent number 20110150875 A1). Using combined immunoprecipitation and Western blotting analyses (Fig. 4), we could only clearly validate the interaction of endogenous GPC3 with PCSK9.
      GPC3 is one of the six members from the glypican family, a subgroup of heparin sulfate proteoglycan family. It is bound to the cell surface of the plasma membrane through a glycosylphosphatidylinositol anchor on its C-terminal domain along with two heparin sulfate side chains (
      • Filmus J.
      • Selleck S.B.
      Glypicans: proteoglycans with a surprise.
      ). In situ hybridization histochemistry revealed that GPC3 mRNA is abundant in many tissues during embryogenesis, including liver primordia (Fig. 14). However, its mRNA expression levels in adult liver are barely detectable (Fig. 14), as reported previously (
      • Hsu H.C.
      • Cheng W.
      • Lai P.L.
      Cloning and expression of a developmentally regulated transcript MXR7 in hepatocellular carcinoma: biological significance and temporospatial distribution.
      ,
      • Capurro M.
      • Wanless I.R.
      • Sherman M.
      • Deboer G.
      • Shi W.
      • Miyoshi E.
      • Filmus J.
      Glypican-3: a novel serum and histochemical marker for hepatocellular carcinoma.
      • Fu S.-J.
      • Qi C.-Y.
      • Xiao W.-K.
      • Li S.-Q.
      • Peng B.-G.
      • Liang L.-J.
      Glypican-3 is a potential prognostic biomarker for hepatocellular carcinoma after curative resection.
      ). Furthermore, GPC3 is overexpressed in 80% of HCCs and correlates with the severity of the tumor (
      • Capurro M.
      • Wanless I.R.
      • Sherman M.
      • Deboer G.
      • Shi W.
      • Miyoshi E.
      • Filmus J.
      Glypican-3: a novel serum and histochemical marker for hepatocellular carcinoma.
      ,
      • Filmus J.
      • Capurro M.
      Glypican-3: a marker and a therapeutic target in hepatocellular carcinoma.
      ). The core protein of 70 kDa with the heparan sulfate side chains contains a recognition motif for furin and PC5/6B (
      • De Cat B.
      • Muyldermans S.-Y.
      • Coomans C.
      • Degeest G.
      • Vanderschueren B.
      • Creemers J.
      • Biemar F.
      • Peers B.
      • David G.
      Processing by proprotein convertases is required for glypican-3 modulation of cell survival, Wnt signaling, and gastrulation movements.
      ). The cleavage of GPC3 at RQYR↓S359 results in the release of a secreted 40-kDa GPC3 N-terminal domain and a cell membrane-associated fragment of 30 kDa (
      • De Cat B.
      • Muyldermans S.-Y.
      • Coomans C.
      • Degeest G.
      • Vanderschueren B.
      • Creemers J.
      • Biemar F.
      • Peers B.
      • David G.
      Processing by proprotein convertases is required for glypican-3 modulation of cell survival, Wnt signaling, and gastrulation movements.
      ). The ∼65-kDa molecular mass of GPC3 observed in the co-IP media with PCSK9 (Fig. 4A) does not correspond to the small N-terminal fragment produced by furin, but it most likely is the shed molecular form of intact GPC3 produced by the C-terminal cleavage of its glycosylphosphatidylinositol anchor by endogenous lipases (
      • Haruyama Y.
      • Kataoka H.
      Glypican-3 is a prognostic factor and an immunotherapeutic target in hepatocellular carcinoma.
      ,
      • Traister A.
      • Shi W.
      • Filmus J.
      Mammalian notum induces the release of glypicans and other GPI-anchored proteins from the cell surface.
      ). GPC3 binds the growth factor Wnt presenting it to its receptor frizzled. Therefore, GPC3 acts as a co-receptor at the cell membrane and promotes the downstream Wnt-frizzled signaling pathway (
      • De Cat B.
      • Muyldermans S.-Y.
      • Coomans C.
      • Degeest G.
      • Vanderschueren B.
      • Creemers J.
      • Biemar F.
      • Peers B.
      • David G.
      Processing by proprotein convertases is required for glypican-3 modulation of cell survival, Wnt signaling, and gastrulation movements.
      ,
      • Capurro M.I.
      • Xiang Y.-Y.
      • Lobe C.
      • Filmus J.
      Glypican-3 promotes the growth of hepatocellular carcinoma by stimulating canonical Wnt signaling.
      ,
      • Gao W.
      • Ho M.
      The role of glypican-3 in regulating Wnt in hepatocellular carcinomas.
      • Capurro M.
      • Martin T.
      • Shi W.
      • Filmus J.
      Glypican-3 binds to Frizzled and plays a direct role in the stimulation of canonical Wnt signaling.
      ). However, other studies have shown that GPC3 can also negatively regulate another pathway by competing for the binding of hedgehog growth factor to its receptor Ptc (
      • Capurro M.I.
      • Xu P.
      • Shi W.
      • Li F.
      • Jia A.
      • Filmus J.
      Glypican-3 inhibits Hedgehog signaling during development by competing with patched for Hedgehog binding.
      ).
      Figure thumbnail gr14
      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.
      Overexpression of GPC3 with PCSK9 and LDLR in Huh7 cells allowed us to demonstrate the interaction of GPC3 with pro-PCSK9 and immature LDLR intracellularly (Fig. 6C), thus likely in the ER as this is the only place where pro-PCSK9 is found (
      • Seidah N.G.
      • Benjannet S.
      • Wickham L.
      • Marcinkiewicz J.
      • Jasmin S.B.
      • Stifani S.
      • Basak A.
      • Prat A.
      • Chretien M.
      The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): liver regeneration and neuronal differentiation.
      ). These observations suggest that GPC3 can interact with the PCSK9-LDLR complex very early in the ER. The co-IP of GPC3, when co-expressed with PCSK9 in Huh7 cells, did not pull down intracellular mature PCSK9 (Fig. 6C) most likely because of PCSK9's rapid exit from the ER leading to its secretion. Nassoury et al. (
      • Nassoury N.
      • Blasiole D.A.
      • Tebon Oler A.
      • Benjannet S.
      • Hamelin J.
      • Poupon V.
      • McPherson P.S.
      • Attie A.D.
      • Prat A.
      • Seidah N.G.
      The cellular trafficking of the secretory proprotein convertase PCSK9 and its dependence on the LDLR.
      ) have described that only the co-expression of PCSK9 fused to the KDEL ER retention sequence could result in the co-immunoprecipitation of immature LDLR with both pro and mature PCSK9 forms.
      Surprisingly, the co-expression of GPC3 with PCSK9 resulted in a decrease in total GPC3 protein levels relative to control in Huh7 cells, suggesting that a putative PCSK9-GPC3 complex could be degraded via an intracellular pathway (Fig. 6B) (
      • Poirier S.
      • Mayer G.
      • Poupon V.
      • McPherson P.S.
      • Desjardins R.
      • Ly K.
      • Asselin M.-C.
      • Day R.
      • Duclos F.J.
      • Witmer M.
      • Parker R.
      • Prat A.
      • Seidah N.G.
      Dissection of the endogenous cellular pathways of PCSK9-induced low density lipoprotein receptor degradation: evidence for an intracellular route.
      ). Because our data showed that overexpression of intracellular membrane-bound GPC3 does not significantly affect the overall intracellular ability of PCSK9 to induce the degradation of the LDLR (Fig. 6A) and that co-expression of LDLR did not change the reduction in total GPC3 protein levels (Fig. 6B), we postulate that PCSK9 can degrade GPC3 in HepG2 cells via an LDLR-independent mechanism. The precise mechanism by which overexpressed PCSK9 can induce the cellular degradation of GPC3 will need further studies, but it was shown that LRP1, an LDLR family member, can mediate the endocytosis and degradation of the hedgehog-GPC3 complex at the cell surface by binding to the heparan sulfate chains of GPC3 (
      • Capurro M.I.
      • Shi W.
      • Filmus J.
      LRP1 mediates Hedgehog-induced endocytosis of the GPC3-Hedgehog complex.
      ). Thus, it is possible that a putative PCSK9-GPC3-LRP1 could be formed, especially because PCSK9 has already been shown to interact and enhance the degradation of LRP1 in HepG2 cells in an LDLR-independent fashion (
      • Canuel M.
      • Sun X.
      • Asselin M.-C.
      • Paramithiotis E.
      • Prat A.
      • Seidah N.G.
      Proprotein convertase subtilisin/kexin type 9 (PCSK9) can mediate degradation of the low density lipoprotein receptor-related protein 1 (LRP-1).
      ).
      To establish a functional role of identified interacting partners on LDL uptake in HepG2 cells, we chose to perform specific mRNA knockdowns (Fig. 7) on all six identified interacting partners, even though they were not all validated in the immunoprecipitation studies (Fig. 4B). Only the mRNA knockdowns of GPC3 and PLTP showed a significant increase of basal DiI-LDL uptake (Fig. 8).
      PLTP knockdown in HepG2 cells increased LDLR mRNA expression and total protein levels (Fig. 9). However, the ability of exogenous hPCSK9 R218S to reduce DiI-LDL uptake (Fig. 11) and protein levels (Fig. 12B) did not change in HepG2 shPLTP versus shNT cells. Taken together, these results clearly demonstrate a role of PLTP on LDLR regulation, but its effect does not involve the modulation of PCSK9 extracellular activity. The PLTP effect on LDLR mRNA results most likely from another indirect pathway because no direct transcriptional function has ever been described from this protein. As our aim was to identify extracellular PCSK9-interacting proteins able to modulate PCSK9 activity toward LDLR degradation, we did not further investigate the role of PLTP on LDLR levels in HepG2 cells. However, it may be interesting to characterize more in detail the cellular function of PLTP on LDLR because studies have focused mostly on its role in HDL metabolism, in the production of apoB-containing lipoproteins, in insulin resistance, or in type 2 diabetes mellitus.
      We also observed that LDLR protein levels were higher upon GPC3 knockdown (shGPC3 cells), although LDLR mRNA levels did not change (Fig. 9). This suggests that GPC3 has post-transcriptional effects on LDLR levels. This hypothesis was supported in DiI-LDL uptake experiments in HepG2 shGPC3 cells using exogenous hPCSK9 R218S, which was nearly three times more efficient to reduce DiI-LDL uptake (Fig. 11) and also more efficient to reduce total LDLR protein levels (Fig. 12A). Based upon these results, we propose that the extracellular interaction between PCSK9 and GPC3 competes with PCSK9 binding to the cell-surface LDLR (Fig. 15). This was confirmed in media swap experiments on HepG2 cells co-expressing GPC3 and LDLR combined with Western blotting analyses of total LDLR protein levels (Fig. 13). As expected, overexpression of GPC3 with LDLR reduces the hPCSK9 WT capacity to degrade the LDLR by 1.6-fold compared with overexpression of LDLR alone in Huh7 cells (Fig. 13). We conclude that GPC3 is not only a novel extracellular and intracellular interactor of PCSK9 but also a competitive extracellular binding partner to the LDLR in HepG2 and Huh7 cell models (Fig. 15). Our study also confirmed that this extracellular binding does not involve PCSK9 CHRD similar to the endogenous inhibitor AnxA2 previously described (
      • Saavedra Y.G.
      • Day R.
      • Seidah N.G.
      The M2 module of the Cys-His-rich domain (CHRD) of PCSK9 protein is needed for the extracellular low density lipoprotein receptor (LDLR) degradation pathway.
      ,
      • Ly K.
      • Saavedra Y.G.
      • Canuel M.
      • Routhier S.
      • Desjardins R.
      • Hamelin J.
      • Mayne J.
      • Lazure C.
      • Seidah N.G.
      • Day R.
      Annexin A2 reduces PCSK9 protein levels via a translational mechanism and interacts with the M1 and M2 domains of PCSK9.
      ). Based on our results, GPC3 interacts with the prodomain and/or catalytic domain of PCSK9 (Fig. 5). It is possible that GPC3 competes with the LDLR to bind the catalytic domain of PCSK9. Further studies will be necessary to validate the precise region or residues implicated in this protein-protein interaction.
      Figure thumbnail gr15
      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 . 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.
      GPC3 is abundantly expressed in mouse (Fig. 14) and human embryonic liver and in human HCC (
      • Hsu H.C.
      • Cheng W.
      • Lai P.L.
      Cloning and expression of a developmentally regulated transcript MXR7 in hepatocellular carcinoma: biological significance and temporospatial distribution.
      ). However, its expression levels in adult human (
      • Hsu H.C.
      • Cheng W.
      • Lai P.L.
      Cloning and expression of a developmentally regulated transcript MXR7 in hepatocellular carcinoma: biological significance and temporospatial distribution.
      ,
      • Iglesias B.V.
      • Centeno G.
      • Pascuccelli H.
      • Ward F.
      • Peters M.G.
      • Filmus J.
      • Puricelli L.
      • de Kier Joffé E.B.
      Expression pattern of glypican-3 (GPC3) during human embryonic and fetal development.
      ) and mouse (Fig. 14) liver is very low, whereas PCSK9 is mainly expressed in human and mouse liver throughout embryogenesis and in the adult. Because our data in Huh7 and HepG2 cells demonstrated that GPC3 can reduce PCSK9 activity to degrade cell-surface LDLR, it is highly plausible that GPC3 might have an important physiological role on the regulation of PCSK9's extracellular activity during embryogenesis, in liver regeneration, or in HCC. Our results also suggest that GPC3 would reduce PCSK9 activity on LDLR degradation (and possibly other PCSK9 targets) during embryogenesis and in certain cancers. It is noteworthy that GPC3 is also very abundant in the small intestine and in the kidney (Fig. 14) where PCSK9 is also significantly expressed (
      • Seidah N.G.
      • Benjannet S.
      • Wickham L.
      • Marcinkiewicz J.
      • Jasmin S.B.
      • Stifani S.
      • Basak A.
      • Prat A.
      • Chretien M.
      The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): liver regeneration and neuronal differentiation.
      ). Because the role of PCSK9 in the small intestine is not yet fully understood, it is difficult to define the impact of GPC3 on PCSK9 activity in this tissue at this time.
      In conclusion, our studies successfully identified a novel and functional interaction between GPC3 and PCSK9. Most importantly, we demonstrate that extracellular GPC3 can act as an endogenous competitive binding partner of PCSK9 to the LDLR in HepG2 and Huh7 cells.

      Author Contributions

      K. L. conducted most of the experiments, analyzed the results, and wrote the manuscript. R. E. performed the experiment on the co-immunoprecipitation of PCSK9-GPC3-LDLR (Fig. 6) and the effect of GPC3 overexpression on PCSK9 extracellular activity in Huh7 cells (Fig. 13). R. Desjardins helped in the production of stable knockdown cell lines. N. G. S. and R. Day conceived and designed the project and wrote the paper with K. L. All authors reviewed the results and approved the final version of the manuscript.

      Acknowledgments

      We thank Jessica Fortin-Mimeault for help with cell line preparation and Jadwiga Marcinkiewicz for the in situ hybridization analyses (Fig. 14).

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