MHC class I antigen presentation of DRiP-derived peptides from a model antigen is not dependent on the AAA ATPase p97

Abstract

CD8(+) T cells are responsible for killing cells of the body that have become infected or oncogenically transformed. In order to do so, effector CD8(+) T cells must recognize their cognate antigenic peptide bound to a MHC class I molecule that has been directly presented by the target cell. Due to the rapid nature of antigen presentation, it is believed that antigenic peptides are derived from a subset of newly synthesized proteins which are degraded almost immediately following synthesis and termed Defective Ribosomal Products or DRiPs. We have recently reported on a bioassay which can distinguish antigen presentation of DRiP substrates from other forms of rapidly degraded proteins and found that poly-ubiquitin chain disassembly may be necessary for efficient DRiP presentation. The AAA ATPase p97 protein is necessary for efficient cross-presentation of antigens on MHC class I molecules and plays an important role in extracting mis-folded proteins from the endoplasmic reticulum. Here, we find that genetic ablation or chemical inhibition of p97 does not diminish DRiP antigen presentation to any great extent nor does it alter the levels of MHC class I molecules on the cell surface, despite our observations that p97 inhibition increased the levels of poly-ubiquitinated proteins in the cell. These data demonstrate that inhibiting poly-ubiquitin chain disassembly alone is insufficient to abolish DRiP presentation.

Figure 1. Transfected EL4/SCRAP cells express elevated levels of p97 protein.

A. EL4/SCRAP cells were transfected with pMSCV containing an IRES insert that allowed for dual expression of Thy1.1 and either wild type (black trace) or a dominant-negative mutant (blue trace) of p97. Cells were stained with anti-Thy1.1 antibody 24 hours post transfected and analyzed by FACS. Representative histograms are shown with the isotype control antibody staining represented by the shaded histogram. B. Total cell lysates from cells transfected with wild type (WT) or dominant negative (DN) p97 vectors were prepared 24 hours after transfection and resolved by SDS-PAGE followed by western blot analysis for p97 protein and actin. C. EL4 cells were transfected with constructs encoding a mutated TCRα- GFP construct and either WT or DN expression plasmids. The following day Thy1.1 cells were analyzed for GFP expression. DN transfected cells had elevated levels of GFP (p<0.05) indicating inhibited ERAD function.

Figure 2. Genetic ablation of p97 does not diminish DRiP antigen presentation.

A. EL4/SCRAP cells were placed in ice-cold citric acid buffer (pH 3) for 2 minutes, washed, re-suspended in tissue culture media, and cultured for 5 hours in the presence of shield-1 before staining with the monoclonal antibody 25D-1.16 to detect K^b-SIINFEKL complexes at the cell surface. K^b-SIINFEKL complexes recovered from near background levels (blue trace) to approximately 50% (orange trace) the level of cells that had not been washed in acid (black trace). EL4 cells that do not express K^b-SIINFEKL are shown as a negative control (shaded histogram) and are considered background 25D- 1.16 staining. B and C. EL4/SCRAP cells were transfected with either an empty vector, wild type (WT) or DN p97 constructs and 24 hours later, washed in mild citric acid to elute existing peptides from MHC class I molecules. Cells were then treated with 5 µM shield-1 and cultured for 5 hours. At indicated times cells were collected and analyzed by FACS. Cells expressing Thy1.1 were subsequently analyzed for GFP expression (B) and the mean fluorescence intensity (MFI) plotted. K^b-SIINFEKL expression (C) was analyzed for both DRiP substrates (left) and the rapidly degraded form of SCRAP (right) and the MFI plotted as a function of time.

Figure 3. Poly-ubiquitinated proteins accumulate in cells expressing DN p97.

Total cell lysates were prepared 24 hours after transfection with empty vector, wild type p97, or DN p97 protein and analyzed by SDS-PAGE and western plot analysis. A. Blots were stained with anti-poly-ubiquitin monoclonal antibodies (top), anti-actin polyclonal antibodies (middle), or anti-p97 monoclonal antibodies (bottom). B. Signals of both poly-ubiquitin conjugated protein were normalized to actin levels in the cell and are depicted graphically. The average ratio of poly-ubiquitin signal to actin for four experiments is plotted. DN p97 expression resulted in a statistically significant accumulation of poly-ubiquitin conjugates in cells (p<0.05).

Figure 4. DBeQ treatment fails to inhibit DRiP antigen presentation.

A. Cells were tested for metabolic turnover of alamarBlue four hours post DBeQ treatment as a proxy for toxicity. Concentrations of DBeQ >1 µM showed single-dose toxic effects. B. DBeQ treatment resulted in accumulation of mutant TCRα-GFP at non-toxic doses of DBeQ (* p<0.05). Acid-washed EL4/SCRAP cells were treated with 5 µM shield-1 and increasing amounts of the p97-inhibiting compound DBeQ or 10 µM MG132. At indicated times, cells were analyzed for either GFP expression (C) and K^b-SIINFEKL accumulation (D).

Figure 5. Inhibition of p97 does not diminish over all levels of cell-surface MHC class I.

A. EL4 cells were transfected with p97 constructs and analyzed 24 hours later for cell-surface K^b expression by FACS by gating onThy1.1 cells. Cells were either left untreated (white bars) or washed in citric acid buffer (black bars) and allowed to recover for five hours before analysis. The staining was done in triplicate. B. EL4 cells were treated with indicated levels of DBeQ for 5 hours and analyzed by FACS for cell-surface K^b expression. No consistent statistical difference at any concentration of DBeQ occurred between experiments, though MG132 did statistically diminish K^b levels (p<0.05). In both experiments, EL4 cells treated with 10 µM MG132 for 5 hours was included as a positive control for inhibiting antigen presentation and diminishing cell-surface K^b.

Figure 6. DBeQ treatment of JY cells does not diminish HLA class I levels.

A. AlamarBlue fluorescence of JY cells treated with either DMSO or increasing doses of DBeQ demonstrates JY cells are more resistant to the single-dose toxic effects of DBeQ. B. JY cells were either washed in citric acid buffer (black bars) or left untreated (white bars) and stained, in triplicate, 5 hours post treatment with indicated concentrations of DBeQ or MG132. Cells were analyzed for total MHC class I expression using the monoclonal antibody W6/32 which detects HLA A,B, and C molecules. This experiment is representative of three independent experiments and no statistical difference between DMSO and DBeQ treated cells was noted.