γ-Glutamyl transpeptidase is a heavily N-glycosylated heterodimer in HepG2 cells

Abstract

The cell surface enzyme γ-glutamyl transpeptidase (GGT) is expressed by human hepatocellular carcinomas (HCCs). HCCs arise from malignant transformation of hepatocytes and are the most common form of primary liver cancer. Identification of tumor-specific, post-translational modifications of GGT may provide novel biomarkers for HCC. The HepG2 cell line, derived from a human HCC, has been used extensively in studies of liver cancer. However, the use of this cell line for studies of GGT have been stymied by reports that HepG2 cells do not process the GGT propeptide into its heterodimeric subunits. The data in this study demonstrate that HepG2 cells do, in fact, produce the mature heterodimeric form of GGT. Immunohistochemical and immunoaffinity analyses provide direct evidence that, in HepG2 cells, GGT is properly localized to the bile canaliculi. Three independent, experimental approaches demonstrate that GGT in HepG2 cells is comprised of two subunits that are more heavily N-glycosylated than GGT from normal human liver tissue. These data directly contradict the dogma in the field. These data support the use of HepG2 cells as a model system for analyzing tumor-specific changes in the post-translational modifications of GGT.

Figure 1

Immunodetection of GGT in HepG2 cells. (A) Localization of GGT in HepG2 cells. GGT activity was localized in acetone-fixed HepG2 cells with a histochemical stain that forms a red product at the site of GGT activity (left panel). Note the bile canalicular structures that have formed between adjacent cells and are identified by high levels of GGT activity. GGT was immunolocalized with the GGT129 antibody in cells fixed in 4% formaldehyde (right panel). Green fluorescence identifies GGT. Blue fluorescence identifies DAPI stained nuclei. The GGT129 antibody also localizes to bile canalicular structures which contain high levels of GGT activity. Scale bars are 10μm. (B and C) Western analyses against the large (B) and small (C) subunits of GGT. Extracts from normal human kidney (lanes B1, C1), liver (lanes B2, C2) and HepG2 cells (lanes B3, C3) were resolved on 10% SDS-PAGE gels and analyzed by western blotting with GGT129 antibody specific to the large subunit of GGT (lanes B1-3) or GGT1/2 H-170 antibody specific to the small subunit of GGT (lanes C1-3). For each blot, equal amounts of GGT activity were loaded per lane. Positions of molecular weight (MW) markers are indicated. Lanes C1and C2 contain a non-specific immunoreactive band at ~ 44kDa.

Figure 2

Contribution of N-glycans to apparent size of the large and small subunits of GGT. (A and B) Extracts from normal human kidney (lanes 1, 5), liver (lanes 2, 6) and HepG2 cells (lanes 3, 7) were heat-denatured and then incubated in the absence (lanes 1-3) or presence (lanes 5-7) of the N-glycosidase, PNGaseF. The extracts were resolved on an 8% (A) or 10% (B) SDS-PAGE gel. Western analyses were carried-out against the large (A) and small (B) subunits of GGT. Positions of molecular weight (MW) markers are indicated. (C) Immunodetection of the large subunit of GGT and Flag-tagged large subunit of GGT in HepG2 cells. Extracts from human liver (lanes 1, 4), mock-transfected HepG2 cells (lanes 2, 5), and Flag-GGT transfected HepG2 cells (lanes 3, 6) were resolved on an 8% SDS-PAGE gel. Western analysis against either the large subunit (L.S) of GGT (lanes 1-3) or the Flag epitope (lanes 4-6) are shown. Arrowhead denotes position of Flag-tagged GGT. Positions of molecular weight (MW) markers are indicated.