Vesicular Integral-Membrane Protein 36 Is Involved in the Selective Secretion of Fucosylated Proteins into Bile Duct-like Structures in HepG2 Cells

Abstract

Fucosylated proteins are widely used as biomarkers of cancer and inflammation. Fucosylated alpha-fetoprotein (AFP-L3) is a specific biomarker for hepatocellular carcinoma. We previously showed that increases in serum AFP-L3 levels depend on increased expression of fucosylation-regulatory genes and abnormal transport of fucosylated proteins in cancer cells. In normal hepatocytes, fucosylated proteins are selectively secreted in the bile duct but not blood. In cases of cancer cells without cellular polarity, this selective secretion system is destroyed. Here, we aimed to identify cargo proteins involved in the selective secretion of fucosylated proteins, such as AFP-L3, into bile duct-like structures in HepG2 hepatoma cells, which have cellular polarity like, in part, normal hepatocytes. α1-6 Fucosyltransferase (FUT8) is a key enzyme to synthesize core fucose and produce AFP-L3. Firstly, we knocked out the FUT8 gene in HepG2 cells and investigated the effects on the secretion of AFP-L3. AFP-L3 accumulated in bile duct-like structures in HepG2 cells, and this phenomenon was diminished by FUT8 knockout, suggesting that HepG2 cells have cargo proteins for AFP-L3. To identify cargo proteins involved in the secretion of fucosylated proteins in HepG2 cells, immunoprecipitation and the proteomic Strep-tag system experiments followed by mass spectrometry analyses were performed. As a result of proteomic analysis, seven kinds of lectin-like molecules were identified, and we selected vesicular integral membrane protein gene VIP36 as a candidate of the cargo protein that interacts with the α1-6 fucosylation (core fucose) on N-glycan according to bibliographical consideration. Expectedly, the knockout of the VIP36 gene in HepG2 cells suppressed the secretion of AFP-L3 and other fucosylated proteins, such as fucosylated alpha-1 antitrypsin, into bile duct-like structures. We propose that VIP36 could be a cargo protein involved in the apical secretion of fucosylated proteins in HepG2 cells.

Keywords: AFP; HepG2; cargo protein; fucosylation; lectin.

Figure 1

Localization of AFP-L3 was observed in the bile duct-like structures in WT HepG2 cells. (A) WT and FUT8-KO HepG2 cells were co-stained with anti-MRP2 (green) and anti-AFP (red) antibodies. (B) WT and FUT8-KO HepG2 cells were co-stained with anti-AFP-L3 (green) and anti-AFP (red) antibodies. Colocalization is displayed in yellow (Merge). Blue is the nuclear stain using 4′,6-diamidino-2-phenylindole (DAPI). The cellular localization of AFP and AFP-L3 is shown in the schematic diagram.

Figure 2

Silver staining of immunoprecipitated products from WT and FUT8-KO HepG2 cells. (A) The silver-stained gel of immunoprecipitated product with anti-AFP antibody using culture supernatants of WT and FUT8-KO HepG2 cells. (B) The silver-stained gel of immunoprecipitated product with anti-AFP antibody using cell lysates of WT and FUT8-KO HepG2 cells. Red arrows indicate bands specifically detected in FUT8 KO samples. Additionally, a red-lined square indicates a group of bands strongly observed in the wild type.

Figure 3

Immunoblots of endosome fractions obtained by ultracentrifugation. FUT8 KO and WT HepG2 cells were subjected to cellular fractionation by ultracentrifugation. The results of immunoblots using anti-LAMP-1, anti-AFP, and anti-AFP-L3 antibodies after electrophoresis are shown. Fractions 3 and 4 were identified as endosome fractions as they colocalized with the endosome marker LAMP-1, and they were used for subsequent immunoprecipitation experiments.

Figure 4

Accumulation of AFP in bile duct-like structures was reduced by VIP36 KO in HepG2 cells. (A) Immunoblot of the expression level of VIP36 in VIP36-KO HepG2 cells compared with WT and FUT8-KO HepG2 cells. (B) Co-staining of WT and VIP36-KO HepG2 cells with anti-MRP2 (green) and anti-AFP (red) antibodies. Colocalization is displayed in yellow (Merge). Blue is the nuclear stain using 4′,6-diamidino-2-phenylindole. (C) Representative images of bile duct-like structures formed on HepG2 cells and stained with anti-AFP (red) and anti-MRP2 (green) antibodies. (D) The percentage of anti-AFP antibody and anti-MRP2 antibody co-positive cells in 30 randomly selected visual fields from images of WT and VIP36-KO HepG2 cells. BC: bile canaliculus (bile duct-like structure). Results are expressed as the mean +/− SD.

Figure 5

Accumulation of alpha-1 antitrypsin in bile duct-like structures was reduced by VIP36 KO in HepG2 cells. (A) Representative images of bile duct-like structures formed on HepG2 cells and stained with anti-alpha-1 antitrypsin (red) and anti-MRP2 (green) antibodies. Histogram shows the percentage of anti-alpha-1 antitrypsin antibody co-positive cells in 30 randomly selected visual fields from images of WT and VIP36-KO HepG2 cells stained with anti-MRP2 antibody. (B) The percentage of anti-alpha-1 antitrypsin antibody and anti-MRP2 antibody co-positive cells in 30 randomly selected visual fields from images of WT and VIP36-KO HepG2 cells. (C) The percentage of anti-alpha-1 antitrypsin antibody and anti-MRP2 antibody co-positive cells in 30 randomly selected visual fields from images of WT, FUT8-KO, and VIP36-KO HepG2 cells. BC: bile canaliculus (bile duct-like structure). Results are expressed as the mean +/− SD. * p < 0.05, ** p < 0.01.