Figure 3: Melan-A/MART-126-35 epitope presentation is ERAD-dependent.
(A) Schematic representation of the Melan-A/MART-126-35 epitope which is entirely localized within the transmembrane domain, except for the first amino acid residue. (B) The melanoma cell line Ma-Mel-91 (Melan-A/MART-1−, HLA-A*0201) was exposed to non-targeting (control) siRNA or siRNA directed against VIMP or HERP1 prior to a subsequent transfection with an expression vector encoding the wild-type full-length Melan-A/MART-1 tumor antigen. The ability of these cells to present the Melan-A/MART-126-35 epitope was evaluated in a 6 h TNF-α release assay upon exposure to Melan-A/MART-126-35 CTL. The content of TNF-α in the supernatants was measured by ELISA. *p < 0.05 vs Ma-Mel-91 cells expressing Melan-A/MART-1 and exposed to control siRNA (Student’s t test) (C) The impact of p97/VCP on Melan-A/MART-126-35 presentation was evaluated by transfecting Ma-Mel-91 cells with Melan-A/MART-1 in combination with either wild-type p97/VCP or a dominant negative mutant of p97/VCP (p97QQ). After 24 h of transfection, the cells were tested for their capacity to present the Melan-A/MART-126-35 antigenic peptide by cultivating them in the presence of Melan-A/MART-126-35 CTL and followed by subsequent measurement of TNF-α by ELISA. In addition, proteasome dependency of Melan-A/MART-126-35 epitope generation was demonstrated by treating the Ma-Mel-91 cells transfected with the Melan-A/MART-1 expression plasmid with 1 μM of the proteasome inhibitor epoxomicin for 4 h. As control, Ma-Mel-91 cells were loaded with the Melan-A/MART-126-35 synthetic peptide or were transfected with the pcDNA3.1 plasmid and loaded with the Melan-A/MART-1 peptide. *p < 0.05 vs Melan-A/MART-1-expressing Ma-Mel-91 cells and ##p < 0.01 vs unloaded Ma-Mel-91 cells (Student’s t test) (D) Silencing of p97/VCP by siRNA results in stabilization of the Melan-A/MART-1 protein expression. HeLa cells were exposed to either non-targeting (control) or p97/VCP siRNA for 24 h prior to a subsequent 24 h transfection with an expression plasmid encoding wild-type Melan-A/MART-1 and subjected to CHX-based chase assay. Cells were collected after 1, 2, 4 and 8 h and Western blot analysis was performed for each time point using antibodies specific for Melan-A/MART-1, p97/VCP and GAPDH (loading control).
