Folding of matrix metalloproteinase-2 prevents endogenous generation of MHC class-I restricted epitope

Abstract

Background: We previously demonstrated that the matrix metalloproteinase-2 (MMP-2) contained an antigenic peptide recognized by a CD8 T cell clone in the HLA-A*0201 context. The presentation of this peptide on class I molecules by human melanoma cells required a cross-presentation mechanism. Surprisingly, the classical endogenous processing pathway did not process this MMP-2 epitope.

Methodology/principal findings: By PCR directed mutagenesis we showed that disruption of a single disulfide bond induced MMP-2 epitope presentation. By Pulse-Chase experiment, we demonstrated that disulfide bonds stabilized MMP-2 and impeded its degradation. Finally, using drugs, we documented that mutated MMP-2 epitope presentation used the proteasome and retrotranslocation complex.

Conclusions/significance: These data appear crucial to us since they established the existence of a new inhibitory mechanism for the generation of a T cell epitope. In spite of MMP-2 classified as a self-antigen, the fact that cross-presentation is the only way to present this MMP-2 epitope underlines the importance to target this type of antigen in immunotherapy protocols.

Figure 1. Various deletions and mutations of MMP-2 cDNA allow MMP-2_560–568 epitope generation by the endogenous pathway in HLA-A*0201 transfected COS-7 cells.

COS-7 cells were cotransfected with HLA-A*0201 plasmid and with (A) plasmids coding for deleted MMP-2 or (B) plasmids coding for mutated MMP-2. 48 h later, M134.12 CTL clones were added to transfected COS-7 cells (E/T ratio 1∶3) and the TNF response was tested after 6 h on Wehi-13 cells. Standard deviations were obtained from duplicates. cDNA NA134-A corresponding to the C-terminal part of MMP-2 contains MMP-2_560-568 epitope and was used as positive control. Transfection efficiency was controlled with GFP transfected COS-7 cells. PS corresponding to the signal sequence (pre), PD corresponding to the prodomaine (pro) and PEX corresponding to the hemopexine domaine of the MMP-2. Data are representative of at least two independent experiments. Error bars indicate standard deviations of duplicates.

Figure 2. Disulfide bond deletion permit MMP-2_560–568 epitope generation by the endogenous pathway in HLA-A*0201+/αvβ3- human tumor cells.

Melanoma cell line M117 and non small cell lung carcinoma line 1355 were transfected with plasmids coding for cystein deleted MMP-2. 48 h later, M134.12 CTL clones were added to tumor cells (E/T ratio 1∶3) and the TNF response was tested after 6 h on wehi-13 cells. Standard deviations were obtained from duplicates. cDNA NA134-A corresponding to the C-terminal part of MMP-2, contains MMP-2_560–568 epitope and was used as positive control. Transfection efficiency was controlled with GFP transfected tumor cells. Data are representative of at least two independent experiments. Error bars indicate standard deviations of duplicates. p<0.005 was considered significant.

Figure 3. Cystein deletion does not impede MMP-2 release.

COS-7 cells transfected with indicated plasmids were cultured for 48 h without FCS. Supernatants were collected and equal amounts of protein were loaded on SDS-PAGE. MMP-2 release was analyzed by (A) gelatin zymographie and (B) western blotting. NT: untransfected COS-7 cells. Data are representative of at least two independent experiments.

Figure 4. Mutated MMP-2 proteins are more rapidly degraded than the wild-type enzyme.

COS-7 cells transfected with indicated plasmids were pulse-labeled with [³⁵S] methionine/cystein for 15 min and chased for 0–24 h. MMP-2 immunoprecipitates were separated by SDS-PAGE and analyzed by autoradiography (A). Data were plotted to indicate the residual protein remaining where the amount of this protein at 0 h time point was calculated to represent 100% of total MMP-2 in each case (B). Data are representative of at least two independent experiments.

Figure 5. MMP-2 endogenous presentation require proteasome and sec61.

A and D: COS-7 cells were cotransfected with plasmids coding for WT MMP-2 or mutated MMP-2 and for HLA-A*0201. 24 h later, COS-7 cells were treated with proteasome inhibitor MG-132 (A) or sec61 inhibitor Exotoxin-A (D). After overnight incubation, cells were washed, counted and cocultured with M134.12 CTL clones (E/T ratio 1∶3). The TNF response was tested 6 h later on wehi-13 cells. B and E: cells treated as in A and D, were pulsed 1 h with peptide before coculture with M134.12. Peptide, corresponding to the MMP-2_560–568 epitope, was used as control of HLA-A*0201 surface expression. C and F: Inhibition percentages are expressed as the percent TNF response of cells treated with drugs versus the percent TNF response of cells untreated, where TNF response of cells untreated was normalized to 100%. Transfection efficiency was controlled with GFP transfected COS-7 cells. Data are representative of two independent experiments. Error bars indicate standard deviations of triplicates.

Figure 6. Hypothetical models for MMP-2_560–568 epitope generation by cross-presentation and by endogenous pathway.

Cross-presentation (1): Newly synthesized wild-type MMP-2 acquire disulfide bonds in the endoplasmic reticulum (ER) before joining the secretory pathway. In the extracellular environment, physiologic activation of the pro-MMP-2 induce the cleavage of the propeptide domain which contains a disulfide bridge (C60-C65: unique to the MMP-2). MMP-2 active form then interact with the integrine αvβ3 and is internalized in clathrin-coated vesicle. Finally MMP-2 is transported to the cytosol, in an unknown mechanism, and degraded by the proteasome. Peptides generated can reach the endogenous pathway (peptides are transported in the ER through TAP, bind to HLA-A*0201 and transported to the cell surface). Endogenous presentation (2): Mutated MMP-2 lacking a disulfide bond can't join the secretory pathway and is retrotranslocated via Sec61. In the cytosol, mutated MMP-2 is degraded by the proteasome and resulting peptides are loaded on MHC class I molecules.