Figure 8.

MT2 cleaves Hfe and Tfr2. A, MT2 cleaves Hfe. HEK293 cells in 12-well plates were co-transfected with constant amounts of pCMV6-Hfe (1.33 μg) and pJB-1-B2M (1.33 μg), increasing amounts of pCMV6-MT2 or S762A-MT2 (0, 0.25, 0.5, 1.0, and 1.33 μg), and decreasing amounts of pEGFP-N1 (1.33, 1.08, 0.83, 0.33, and 0 μg). After 48 h of transfection, Hfe and MT2 in ∼150 μg of cell lysate, proteins were immunodetected by using anti-FLAG antibody, and β-actin and EGFP by specific antibodies. HEK293 cells (Ctrl) were included as a negative control. Two Hfe images with different exposure times are presented. B, MT2 decreases cell-surface Hfe. HEK293 cells were co-transfected with pCMV6-Hfe, pJB-1-B2M, and pEGFP-N1 or pCMV6-MT2 or S762A-MT2 at 1:1:1 ratios of plasmid DNA. Biotinylation of cell-surface proteins and immunodetection were performed essentially the same as described in the legend to Fig. 6C. Hfe was detected by using an anti-FLAG antibody. C and D, MT2 cleaves Tfr2. Co-transfection of HEK293 cells with pcDNA3-Tfr2 and pEGFP-N1, pCMV6-MT2, or S762A-MT2, biotinylation of cell-surface proteins, and immunodetection were performed essentially the same as described in the legend to Fig. 6, B and C. At about 24 h post-transfection, medium was changed to Opti-MEM, 1% FCS. Analysis was performed after another 24 h of incubation. About 600 μl of CM was collected for analysis of Tfr2 release. Tfr2 was detected by using a rabbit antibody against mouse Tfr2 extracellular domain. E, qRT-PCR analysis of MT2 mRNA in HepG2 cells and HepG2 cells stably expressing transfected-human MT2 (HepG2-MT2). Results are expressed as the amount relative to that of β-actin. n = 3 biological replicates. F, leupeptin inhibits the cleavage of HJV by endogenous MT2 in HepG2 cells. HepG2 cells stably expressing transfected-HJV (HepG2-HJV) were incubated in Opti-MEM, 1% FCS with or without 100 μm leupeptin (Leu) for about 18 h. Cell lysate (L) and CM were collected for immunodetection of HJV by an anti-HJV antibody. β-Actin was used as a loading control. In CM, the upper and lower HJV bands corresponded to the cleavage products by furin and MT2, respectively. G, leupeptin inhibits MT2 cleavage of endogenous TfR2 in HepG2 cells. HepG2 and HepG2-MT2 cells were incubated in Opti-MEM, 1% FCS with or without 100 μm leupeptin (Leu) for about 18 h. CM and cell lysate were collected. TfR2 in CM was immunoprecipitated by a mouse anti-TfR2 monoclonal antibody, and immunodetected by a rabbit anti-TfR2 antibody. About 5% of cell lysate (∼150 μg of proteins) was used for immunodetection of MT2, TfR2, and β-actin. All experiments were repeated at least three times (technical replicate = 1 or 2; independent biological replicates ≥ 3) with consistent results.