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Comparative Study
. 2013 Aug 20;8(8):e71859.
doi: 10.1371/journal.pone.0071859. eCollection 2013.

Comparative Proteomics Reveals Novel Components at the Plasma Membrane of Differentiated HepaRG Cells and Different Distribution in Hepatocyte- And Biliary-Like Cells

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Free PMC article
Comparative Study

Comparative Proteomics Reveals Novel Components at the Plasma Membrane of Differentiated HepaRG Cells and Different Distribution in Hepatocyte- And Biliary-Like Cells

Catalina Petrareanu et al. PLoS One. .
Free PMC article

Abstract

Hepatitis B virus (HBV) is a human pathogen causing severe liver disease and eventually death. Despite important progress in deciphering HBV internalization, the early virus-cell interactions leading to infection are not known. HepaRG is a human bipotent liver cell line bearing the unique ability to differentiate towards a mixture of hepatocyte- and biliary-like cells. In addition to expressing metabolic functions normally found in liver, differentiated HepaRG cells support HBV infection in vitro, thus resembling cultured primary hepatocytes more than other hepatoma cells. Therefore, extensive characterization of the plasma membrane proteome from HepaRG cells would allow the identification of new cellular factors potentially involved in infection. Here we analyzed the plasma membranes of non-differentiated and differentiated HepaRG cells using nanoliquid chromatography-tandem mass spectrometry to identify the differences between the proteomes and the changes that lead to differentiation of these cells. We followed up on differentially-regulated proteins in hepatocytes- and biliary-like cells, focusing on Cathepsins D and K, Cyclophilin A, Annexin 1/A1, PDI and PDI A4/ERp72. Major differences between the two proteomes were found, including differentially regulated proteins, protein-protein interactions and intracellular localizations following differentiation. The results advance our current understanding of HepaRG differentiation and the unique properties of these cells.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Validation of the plasma membrane (PM) purification.
Equal amounts of proteins from total HepaRG cell lysate (TL) and purified PM fraction were loaded on SDS-PAGE and analyzed by Western blotting using organelle-specific Abs. The molecular weight markers are indicated. The representative blot of three independent experiments is shown.
Figure 2
Figure 2. Summary of the proteomic analysis of the PM from undifferentiated (ND) and differentiated (D) HepaRG cells.
(A and B) SDS-PAGE resolved proteins were analyzed by LC-MS/MS as described. Venn diagrams show the number of proteins identified in PM from (ND) and (D) HepaRG cells in experiments 1 (A) and 2 (B).
Figure 3
Figure 3. Classification of the proteins identified at the PM of (ND) and (D) HepaRG cells.
The mascot search results from experiments 1 and 2 were exported as.dat files and processed with the license-based Scaffold 4.0 software (www. proteomescience.com). The proteins identified in (ND) and (D) cells were classified according to their involvement in a biological process (A), subcellular localization (B) and molecular function (C).
Figure 4
Figure 4. Analysis of differentially expressed proteins identified at the PM of (ND) and (D) HepaRG cells.
The PM fractions of (ND) and (D) cells were analyzed by Western blotting using Abs specific for proteins with significantly altered levels of expression following differentiation. The molecular weight markers are indicated.
Figure 5
Figure 5. Identification and relative quantification of Endoplasmin, Cyclophilin A and Cathepsin D.
(A) A doubly-charged peak at m/z of 594.56 (D) was fragmented by MS/MS and produced a series of b and y peaks shown in the MS/MS spectrum leading to identification of the Endoplasmin-derived SILFVPTSAPR peptide. (B) A double-charged peak at m/z of 973.31 (D) was fragmented by MS/MS and produced a series of b and y peaks shown in the MS/MS spectrum leading to identification of the Cyclophilin A-derived VNPTVFFDIAVDGEPLGR peptide. (C) A double-charged peak at m/z of 628.53 (D) was fragmented by MS/MS and produced a series of b and y peaks shown in the MS/MS spectrum leading to identification of the Cathepsin D-derived FDGILGmAYPR sequence (m denoted oxidized methionine. (D) Comparison of the intensities of MS spectra for the doubly charged precursor peaks with m/z of 594.58, 973.34 and 628.56 corresponding to peptides derived from Endoplasmin (A), Cyclophilin A (B) and Cathepsin D (C). The intensity scale for the spectra from both (D) (upper panels) and (ND) cells (lower panels), for each individual peptide was identical.
Figure 6
Figure 6. Analysis of differentially expressed proteins in hepatocyte- and biliary-like HepaRG cells (I).
Cells were seeded on coverslips in 6-well plates and either differentiated (D) or maintained untreated, as control (ND). Expression of Annexin A1 (panel a) and Cyclophilin A (panel b) in hepatic (H) and biliary (B) cells was evidenced using specific Abs. Albumin expression was also investigated as a marker for differentiation to adult hepatocytes (panels a and b). Samples were analyzed with a Zeiss LSM 710 confocal microscope following mounting with Vectashield Mounting Medium containing DAPI, to visualize the nuclei (blue). Images were taken with the 63x objective and processed with ZEN software. Scale bar is 20 µm.
Figure 7
Figure 7. Analysis of differentially expressed proteins in hepatocyte- and biliary-like HepaRG cells (II).
HepaRG cells were analyzed as in Figure 4, except that expression of PDI (panel a), PDI A4 (panel b), Cathepsin D (panel C) and Cathepsin K (panel d) were evidenced using specific Abs. Where possible, albumin expression was also evidenced (panels b and d).

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Grant support

The work was supported by the Romanian Academy project 3 of the Institute of Biochemistry and POSDRU/89/1.5/S/60746; Catalina Petrareanu was supported by the Sectoral Operational Programme Human Resources Development 2007–2013 of the Romanian Ministry of Labour, Family and Social Protection through the Financial Agreement POSDRU/107/1.5/S/76903 grant. This work was also supported in part by the Army Research Office through the Defense University Research Instrumentation Program (DURIP grant #W911NF-11-1-0304 to CCD). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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