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

Kévin Ly,Rachid Essalmani,Roxane Desjardins,Nabil G Seidah,Robert Day

doi: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.

Highlights

Hypercholesterolemia, characterized by elevated plasma levels of low density lipoprotein (LDL)-cholesterol (LDL-C),3 is proprotein convertase subtilisin/kexin type 9 (PCSK9) is mostly expressed and secreted into the plasma from the liver and is found at lower levels in small intestine and kidneys [2, 3]
Proteomic Analysis from PCSK9 Immunoprecipitation—To probe for new interaction partners of PCSK9, we performed an immunoprecipitation of PCSK9 (PCSK9-IP) on conditioned media produced from HepG2 cells stably overexpressing V5-tagged PCSK9 (HepG2 pIR/hPCSK9-V5) [27] followed by UHPLC-mass spectrometry (MS)/MS analysis
The zymogen prohPCSK9-V5 form is observed at ϳ75 kDa and the autocatalytically processed mature form at ϳ65 kDa in the cell lysate, whereas the mature form is only seen in the conditioned media (Fig. 1B), as reported previously [2, 4]

Summary

Introduction

Hypercholesterolemia, characterized by elevated plasma levels of low density lipoprotein (LDL)-cholesterol (LDL-C), is PCSK9 is mostly expressed and secreted into the plasma from the liver and is found at lower levels in small intestine and kidneys [2, 3]. The role of PCSK9 in cholesterol metabolism derived from its ability to bind the EGF-A domain of the LDLR [6] either intracellularly or extracellularly [7], and the PCSK9-LDLR complex is sorted to the endosome/lysosome pathway for degradation [8]. This action reduces LDLR protein levels at the cell surface and decreases the elimination of circulating LDL-C. We sought to identify novel extracellular interaction partners that could participate and regulate the PCSK9-LDLR complex formation and LDLR degradation

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Results

Conclusion