Using intein catalysis to probe the origin of major histocompatibility complex class I-presented peptides

Abstract

All vertebrate nucleated cells generate peptides from their expressed gene products and then display them at the cell surface bound to MHC class I molecules. This allows CD8(+) T cells to detect and eliminate abnormal cells that are synthesizing foreign proteins, e.g., from viruses or mutations. To permit the immune system to more uniformly monitor a cell's proteins, regardless of their half-life or location, it has been thought that the products of rapid degradation of the mistakes of protein synthesis (defective ribosomal products, DRiPs) preferentially contribute to the class I-presented peptides. However, using intein catalysis to generate peptide sequences exclusively by posttranslational splicing of mature proteins, we show here that presented peptides can be generated from fully folded and functional proteins. Remarkably, the presentation of peptides from two model mature proteins is just as efficient as from newly synthesized proteins subject to errors in translation or folding. These results indicate that for the constructs we have analyzed, DRiPs are not a more efficient source of class I peptides for antigen presentation than the turnover of mature functional proteins. Accordingly, our data suggest that one of the major ways the immune system evaluates the health of cells is by monitoring the breakdown products of the proteome.

Fig. 1.

Intein structure–function and construct description. (A) Clustal W alignment of the amino acid sequences of the M. tuberculosis and P. chrysogenum mini-inteins used. Regions involved in the splicing catalysis are highlighted in black. (B) Diagram of the crystal structure of the M. tuberculosis full-length RecA intein (Protein Data Bank ID code 2IMZ). Highlighted in black are the terminal residues and the histidine side chain in the N3 motif, all of which are involved in catalysis (27). (C–F) Schematic of lentiviral construct inserts (note that the product of the prespliced construct is the same as generated by intein splicing). Positions of the class I epitopes SIINFEKL (S8L), KCSRNRQYL (K9L), and ASNENMETM (A9M) are shown relative to GFP, HA tag, and the two inteins.

Fig. 2.

Intein splicing kinetics. (A) Autoradiogram after SDS/PAGE and anti-HA immunoprecipitation of lysates from GFP-RecA-HY lentivirus-infected E36 cells pretreated with epoxomicin (5 μM, 2 h) subjected to [³⁵S]-Met/Cys pulse label followed by a “cold” chase. Unspliced intein is indicated with a black arrowhead, and the spliced version with an open arrowhead. (B) Same as A except using lysates from GFP-PRP-NP lentivirus-infected E36 cells. (C and D) Arrows as in A, densitometry quantification of the bands on the autoradiograms above. See also Figs. S1, S3, and S4.

Fig. 3.

Presentation at steady state from A9M constructs. E36 cells transduced with lentiviral doxycyline (Dox)-inducible GFP-NP (prespliced), GFP-PRP-NP (intein), or N3 (splicing defective). GFP-PRP-NP mutant constructs were induced with the indicated amounts of Dox and the following measurements were performed in A–D: (A) GFP mean fluorescence intensity (MFI) after 24 h. (B) Cell surface presentation of D^b-A9M complexes measured by coculture in the presence of brefeldin A (to stop further transport of MHC class I complexes) of 12.64-CD8αβ-Luc T-cell hybrids (35) with E36 cells induced for 24 h. (C) D^b-A9M complexes assayed as in B but with 6 h GFP-PRP-NP E36 Dox induction in the presence or absence of epoxomicin (5 μM). (D) Cell surface presentation of K^b-S8L complexes at 24 h postinduction was measured with the 25-D1.16 antibody (39). Data from A–C were replotted as follows: (E) Presentation of K^b-S8L epitope relative to GFP MFI. (F) Presentation of D^b-A9M epitope relative to GFP MFI. (G) Linear regression of presentation of D^b-A9M versus K^b-S8L epitope for the various constructs.

Fig. 4.

Presentation at steady-state from K9L constructs. Similar to Fig. 3, except the E36 cells were transduced with constructs carrying GFP-K9L (prespliced), GFP-RecA-K9L (intein), or N3 (splicing defective) mutant GFP-RecA-HY and the D^b-K9L epitope was measured instead of D^b-A9M using HY TCR-transgenic T cells: (A) GFP mean fluorescence intensity (MFI) after 24 h. (B) Cell surface presentation of D^b-K9L complexes quantified by intracellular cytokine staining assays for TNFα production by coculture in the presence of brefeldin A (to stop further transport of MHC class I complexes) of HY TCR-transgenic T cells with E36 cells induced for 24 h. (C) D^b-K9L complexes assayed as in B but with 4 h GFP-RecA-HY E36 Dox induction in the presence or absence of epoxomicin (5 μM). (D) Cell surface presentation of K^b-S8L complexes at 24 h postinduction, quantified by assaying the production of luciferase from RF33-Luc cells (35) cocultured with the E36 cells for 16 h in the presence of brefeldin A (1 μg/mL). Data from A–C were replotted as follows: (E) Presentation of K^b-S8L epitope relative to GFP MFI. (F) Presentation of D^b-K9L epitope relative to GFP MFI. (G) Linear regression of presentation of D^b-K9L versus K^b-S8L epitope.

Fig. 5.

Time course of epitope presentation. (A) Same as Fig. 4G, except presentation was measured at 2, 4, 12, and 24 h. (B) Same as Fig. 3G, except presentation was measured at 3, 6, 12, and 24 h.

Fig. 6.

Model and comparison between predictions and observations. (A) Diagram of the processes modeled in silico (Materials and Methods). (B and C) Presentation from cells expressing the prespliced vs. the intein constructs (GFP-RecA-HY and GFP-PRP-NP, respectively) as a function of Dox induction time. Observed points were calculated as the quotient of the slopes of the regression lines fitted in Fig. 5 A and B and plotted as mean ± SEM of at least three independent experiments. Predicted curves were obtained from a KinTek Explorer (41) simulation experiment (Materials and Methods, Table S1, and Fig. S3).