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

Edited by Xiao-Fan WangThe 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.

The cargo receptor SURF4 promotes the efficient cellular secretion of PCSK9

“…Previous investigation of the ER cargo receptor LMAN1 demonstrated no specificity for SEC24A over other SEC24 paralogs, making this unlikely to serve as a PCSK9 cargo receptor ( Wendeler et al, 2007 ). Earlier analyses of PCSK9-interacting proteins ( Ly et al, 2016 ; Xu et al, 2012 ; Denis et al, 2011 ) did not identify a clear receptor mediating PCSK9 secretion. Here, we developed a novel strategy for ER cargo receptor identification that combines proximity-dependent biotinylation with CRISPR-mediated functional genomic screening.…”

The cargo receptor SURF4 promotes the efficient cellular secretion of PCSK9

“…Previous investigation of the ER cargo receptor LMAN1 demonstrated no specificity for SEC24A over other SEC24 paralogs, making this unlikely to serve as a PCSK9 cargo receptor 10 . Earlier analyses of PCSK9-interacting proteins [11][12][13] did not identify a clear receptor mediating PCSK9 secretion. Here, we developed a novel strategy for ER cargo receptor identification that combines proximity-dependent biotinylation with CRISPR-mediated functional genomic screening.…”

Identification of amino acid residues in the ligand binding repeats of LDL receptor important for PCSK9 binding

“…However, it is also possible that the LRs, like the YWTD domain shown in the crystal structure (20), may have a minor interaction with PCSK9. Alternatively, several PCSK9 potential binding partners have been reported (14, 43, 44). It will be of interest to investigate whether the LRs of LDLR, especially D172 and D203, are involved in the interaction with these potential partners.…”