A major fraction of cytosolic post proline peptidase activity is contributed by DPP9. A, left, 5 μg of cytosol from HeLa cells were tested for the cleavage of R-AMC, GP-AMC, and AAF-AMC. Reactions were performed for the indicated times, and fluorescence was measured using 380-nm (excitation) and 450-nm (emission) filters. Right, 5 μg of HeLa cell cytosol were treated with the indicated concentrations of a specific DPP8/9 inhibitor (compound 2) before the addition of 250 μm GP-AMC. B, HeLa cells were transfected with siRNA against POP, DPP8, DPP9, or control siRNA and harvested after 72 h. Shown is a Western blot of cytosol extracts from the siRNA treated cells (10 μg of protein extract per lane) developed with antibodies against DPP8, DPP9, POP and tubulin as a loading control. Cytosol fractions from silenced cells were then tested for release of AMC from GP-AMC, KP-AMC, VP-AMC, WP-AMC, and DP-AMC as in A (5 μg of cytosolic fractions were used per reaction). C, cells were transfected with three different DPP9 siRNAs or control siRNA and analyzed for activity against GP-AMC as in A. A Western blot is shown to confirm the down-regulation of DPP9 in the analyzed fractions, developed with antibodies against DPP9 and tubulin. All kinetic assays (A–C) were repeated at least three times in triplicate; shown are results of one triplicate assay. D, to estimate DPP8 and DPP9 protein levels at steady state, three different concentrations of HeLa total cell lysates (5, 10, or 20 μg) and defined concentrations of recombinant (rec) DPP8 (1, 5, 10, 20 ng) or DPP9 (5, 10, 20, 40 ng) purified from E. coli were compared by immunoblotting with DPP8- or DPP9-specific antibodies.

Antigens containing proline at position 2 are competitive substrates for DPP9. GP-AMC (100 μm) was mixed with a competing peptide (400 μm) before the addition of 50 nm recombinant DPP9. AMC release was measured after 5 min using 380 nm (excitation) and 450 nm (emission) filters. Sequence of control peptides 1 and 2 are EQKLISEEDL and DYKDDDDK, respectively. K_i values were calculated using detailed Michaelis-Menten analysis in competition assays with GP-AMC (by mixing 0, 100, or 500 μm peptide with varying concentrations of GP-AMC) using the Prism software. Each experiment was repeated at least four times (each in triplicate); shown are the results of one experiment done in triplicate.

Administration of a DPP8/9-specific inhibitor to cells leads to increased presentation of the RU1_34–42 antigen. A, 293-EBNA cells were transiently transfected with plasmids expressing RU1 and HLA-B51. 48 h later cells were treated for 2 h with either 1 μm concentrations of the proteasome inhibitor epoxomicin (Epoxo) or 100 μm DPP8/9 specific inhibitor (m. DPP8/9), which was added to the medium. Alternatively, cells were electroporated with 100 μm DPP8/9 inhibitor (e. DPP8/9) or DMSO for control. Two hours later CTL clone 381/84 was added to the cells, and INF-γ release was measured (black bars, right graph). Saturating amounts of the antigenic peptide were added to cells before incubation with CTL clone 381/84 (white bars, right graph) to assess their viability. Shown on the left side is a simplified presentation of the inhibitor effect on the peptide presentation. It consists of the ratio between INF-γ release from tested cells (black bars, right graph) divided by INF-γ release from the control cells that were incubated with saturating amounts of the peptide before the assay (white bars, right graph). Each experiment was repeated at least three times, each time in triplicate (shown is one experiment in triplicate). The error bars show S.D. of the triplicates. B, cells in A were tested for their capacity to present MAGE-A3_168–176 to HLA-A1 using CTL clone A10. CTL clone A10 was isolated from a HLA-A1 melanoma patient vaccinated with a recombinant pox virus encoding MAGE-A3168–176 C, lysates of cells in A (5 μg) were tested for activity against 5 μm GP-AMC, 50 μm succinyl-LLVY-AMC, and 5 μm R-AMC. Each assay was repeated three times.

DPP9 is a rate-limiting enzyme that contributes both positively and negatively to the repertoire of peptides presented by MHC class I. Ubiquitinated proteins are degraded by the proteasome to peptides of varying length. N-terminal-extended versions of peptide antigens are among the many different peptides produced by proteasomal degradation (antigenic peptides are presented as gray boxes, and N-terminal extensions are presented as white boxes). These extensions are subsequently removed by endo- and aminopeptidases to produce the mature epitope. A, antigenic peptides such as RU1 contain a XaaPro at the N terminus and can be destroyed by DPP9 if they are present in the cytosol. In this case silencing of DPP9 results in increased presentation of the mature epitope. B, other antigenic peptides may depend on DPP9 for presentation if a XaaPro dipeptide flanks the N terminus of the mature antigen.

In vitro processing of XP-AMC substrates by recombinant DPP9. Left, Coomassie staining of recombinant DPP9, expressed and purified from E. coli. The graph shows the results of a Michaelis-Menten assay performed as a triplicate assay using 50 nm DPP9 and varying concentrations of the XP-AMC substrate. The table shows calculated k_cat, K_m, and k_cat/K_m results for different substrates. Values were calculated using non-linear regression (Prism software).

Recombinant DPP8 and DPP9 share similar substrate specificity. Two peptide libraries were tested in competition assays with GP-AMC, XPYGSFKHV and VPXGSFKHV, where X is one of 20 amino acids. GP-AMC (100 μm) was mixed with a competing peptide (400 μm) before the addition of 50 nm recombinant DPP8 or DPP9. AMC release was calculated 5 min after initiation of reaction. Relative AMC release was calculated by setting AMC release in the absence of competing peptide to 100%. A, competition assays for DPP9 with peptide library XPYGSFKHV. B, competition assays for DPP8 with peptide library XPYGSFKHV. C, competition assays for DPP9 with peptide library VPXGSFKHV. Each experiment was performed at least four times, each in triplicate. Shown are the results of a triplicate.

The RU1_34–42 antigen is a substrate for DPP9 in vivo. A, BB64-RCC and 293-EBNA cells were transfected with siRNA against DPP9 or against GFP as control. 24 h later 293-EBNA cells were transiently transfected with plasmids expressing RU1 and HLA-B51 (BB64-RCC express RU1 and HLAB51 endogenously). CTL clone 381/84 was added to the cells, and INF-γ release was measured (black bars, right graph). As a control, saturating amounts of the antigenic peptide were added to cells before incubation with CTL clone 381/84 (white bars, right graph) to assess the viability after siRNA treatment. Shown on the left side is a simplified presentation of the siRNA effect on the peptide presentation. It consists of the ratio between INF-γ release from tested cells (black bars, right graph) divided by INF-γ release from the control cells that were incubated with the peptide before the assay (white bars, right graph). Each experiment was repeated at least three times, each time in triplicate (shown is one experiment in triplicate). The error bars show S.D. of the triplicates. Shown is a Western blot analysis of the cells used as targets in the CTL assay. β-Actin was used as a loading control. B, same experiment as in A, except that cells were transfected with siRNA against DPP8 or GFP for control.