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

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 LDI, 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.