Two abundant proteasome subtypes that uniquely process some antigens presented by HLA class I molecules - PubMed (original) (raw)

Two abundant proteasome subtypes that uniquely process some antigens presented by HLA class I molecules

Benoît Guillaume et al. Proc Natl Acad Sci U S A. 2010.

Abstract

Most antigenic peptides presented by MHC class I molecules result from the degradation of intracellular proteins by the proteasome. In lymphoid tissues and cells exposed to IFNγ, the standard proteasome is replaced by the immunoproteasome, in which all of the standard catalytic subunits β1, β2, and β5 are replaced by their inducible counterparts β1i, β2i, and β5i, which have different cleavage specificities. The immunoproteasome thereby shapes the repertoire of antigenic peptides. The existence of additional forms of proteasomes bearing a mixed assortment of standard and inducible catalytic subunits has been suggested. Using a new set of unique subunit-specific antibodies, we have now isolated, quantified, and characterized human proteasomes that are intermediate between the standard proteasome and the immunoproteasome. They contain only one (β5i) or two (β1i and β5i) of the three inducible catalytic subunits of the immunoproteasome. These intermediate proteasomes represent between one-third and one-half of the proteasome content of human liver, colon, small intestine, and kidney. They are also present in human tumor cells and dendritic cells. We identified two tumor antigens of clinical interest that are processed exclusively either by intermediate proteasomes β5i (MAGE-A3(271-279)) or by intermediate proteasomes β1i-β5i (MAGE-A10(254-262)). The existence of these intermediate proteasomes broadens the repertoire of antigens presented to CD8 T cells and implies that the antigens presented by a given cell depend on their proteasome content.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Identification of intermediate proteasomes in human cancer cell lines. (A) Immunoblots performed with total proteasomes isolated by immunoprecipitation with anti-α2 mAb MCP21 (39) from tumor lines not exposed to IFNγ. Subunits were detected using the rabbit antibodies characterized in

Figs. S2

–

S4

. (B) Characterization of intermediate proteasomes form melanoma line EB81-MEL. Cell lysates were depleted of β5i-containing or β5-containing proteasomes before immunoprecipitation with mAb MCP21 of all of the remaining proteasomes and their analysis by immunoblot. Where indicated, a second depletion with anti-β1i was performed after the β5 depletion. The commercial anti-β1 antibodies used were of mouse origin and therefore in some cases the secondary antibody cross-reacted with the light chain of the mouse mAb used for the immunoprecipitation (Upper bands). The control showing complete depletion of β5-containing proteasomes is shown on

Fig. S8

.

Fig. 2.

Fig. 2.

Exclusive processing of peptide MAGE-A10254–262 by the intermediate proteasome β1i-β5i. (A) Processing of peptide GLYDGMEHL254–262 by 293-EBNA cells containing different proteasome subtypes (characterized in

Fig. S2

). 293-SP, untransfected 293-EBNA cells containing only standard proteasomes (SP); 293-β1i, 293-EBNA cells transfected with a construct encoding β1i; 293-β1i-β5i, transfected with β1i and β5i; 293-IP, transfected with β1i, β2i, and β5i, and containing only immunoproteasomes (IP). Cells were transfected with an HLA-A2 construct and the indicated amount of plasmid encoding MAGE-A10. As positive control, cells were loaded with the synthetic peptide at 0.5 μg/mL MAGE-A10–specific CTL was added and TNF production was measured. (B) Detection of antigenic peptide GLYDGMEHL in digests obtained by incubating precursor peptide ALNMMGLYDGMEHLIYGEPRKLLT with purified 20S proteasomes. Digests were analyzed by mass spectrometry coupled to HPLC, and the detection of the relevant ion was plotted as a function of the degradation of the precursor peptide. AU, arbitrary units. (C) MS detection of the indicated peptide fragments in the digests shown in B, analyzed at a time point corresponding to a precursor degradation of 59% (β5i), 55% (β1i-β5i), 53% (SP), and 40% (IP).

Fig. 3.

Fig. 3.

Exclusive processing of peptide MAGE-A3271–279 by the intermediate proteasome β5i. (A) Restoration of the presentation of MAGE-A3 peptide FLWGPRALV271–279 by tumor cells after treatment with proteasome inhibitors. Melanoma SK23-MEL and myeloma L363 were treated with lactacystin (50 μM), epoxomicin (1 μM), or PS341 (50 nM) for 1 h before addition of the MAGE-A3271–279–specific CTL. The synthetic peptide (0.2 μg/mL) was loaded on tumor cells as positive control. Production of TNF was measured after 24 h. (B) Processing of peptide FLWGPRALV271–279 by 293-EBNA cells containing different proteasome subtypes. Cells were transfected with HLA-A2 and the indicated amount of plasmid encoding MAGE-A3. As positive control, cells were loaded with the peptide at 1 μg/mL. MAGE-A3–specific CTL was added and TNF production was measured. (C) Detection of antigenic peptide FLWGPRALV in digests obtained by incubating precursor peptide SPDACYEFLWGPRALVETSYVKV with purified 20S proteasomes, in the absence (solid lines) or in the presence (dotted lines) of 1 μM epoxomicin. (Left) Digests were analyzed by mass spectrometry coupled to HPLC, and the detection of the relevant ion was plotted as a function of the degradation of the precursor peptide. (Right) The same digests were loaded onto HLA-A2+ melanoma cells LB2667-MEL. MAGE-A3–specific CTL was added and TNF production was measured. Similar results were obtained when lactacystin was used instead of epoxomicin. (D) MS detection of the indicated peptide fragments in the digests shown in C, analyzed at a time point corresponding to a precursor degradation of 41% (β5i), 36% (β5i + epoxo), 35% (SP), 41% (SP + epoxo), 22% (β1i-β5i), 28% (β1i-β5i + epoxo), 28% (IP), and 26% (IP + epoxo). Similar results were obtained when lactacystin was used instead of epoxomicin.

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