26S proteasomes and immunoproteasomes produce mainly N-extended versions of an antigenic peptide

Abstract

Protein degradation by proteasomes is the source of most antigenic peptides presented on MHC class I molecules. To determine whether proteasomes generate these peptides directly or longer precursors, we developed new methods to measure the ef®ciency with which 26S and 20S particles, during degradation of a protein, generate the presented epitope or potential precursors. Breakdown of ovalbumin by the 26S and 20S proteasomes yielded the immunodominant peptide SIINFEKL, but produced primarily variants containing 1±7 additional N-terminal residues. Only 6±8% of the times that ovalbumin molecules were digested was a SIINFEKL or an N-extended version produced. Surprisingly, immunoproteasomes which contain the interferon-g-induced b-subunits and are more ef®cient in antigen presentation, produced no more SIINFEKL than proteasomes. However, the immunoproteasomes released 2±4 times more of certain N-extended versions. These observations show that the changes in cleavage speci®city of immunoproteasomes in¯uence not only the C-terminus, but also the N-terminus of potential antigenic peptides, and suggest that most MHC-presented peptides result from N-terminal trimming of larger proteasome products by aminopeptidases (e.g. the interferon-ginduced enzyme leucine aminopeptidase).

Bioinformatic analysis of functional differences between the immunoproteasome and the constitutive proteasome

“…Therefore, it is remarkable that when enolase was degraded with immunoproteasomes and constitutive proteasomes, approximately the same number of cleavage sites were observed (similar results were obtained for degradation of ovalalbumin Cascio et al 2001). One reason for this is that the immunoproteasome uses leucine, which is a very abundant amino acid, much more frequently than the constitutive proteasome.…”

“…The cleavage occurs between the P1 and the P1 0 position (Berger and Schechter 1970). It has been suggested that the P1 position is the most important position determining cleavage (Altuvia and Margalit 2000;Cascio et al 2001), although the flanking region may also be important (Ossendorp et al 1996;Beekman et al 2000;Mo et al 2000). One way of analyzing such data is to calculate the information content at the cleavage site and in the region flanking the cleavage site.…”

“…However, we do find a significant difference between the percentages of MHC ligands that can be produced by the immunoproteasome and the constitutive proteasome. (Cascio et al 2001) also found that peptides containing the dominant ovalalbumin epitope (SIIN-FEKL) are generated significantly more often by the immunoproteasomes than the constitutive proteasomes despite the fact that the average length of ovalalbumin degradation products was the same for the immunoproteasome and the constitutive proteasome.…”

“…Similar distributions are obtained for human proteins. Often peptides are produced by the proteasome with an extension on their amino-termini (Cascio et al 2001). Aminopeptidases in the cytosol or in the endoplasmic reticulum trim the Nterminal of these peptides (Stoltze et al 2000b).…”

“…This co-evolution is limited to the specificity of C-terminal of MHC ligands and the specificity of the P1 position in the immunoproteasome digests. The P1 0 and P2 0 positions do not become the N-terminal of MHC ligands, due to additional trimming that takes place in the cytoplasm and the endoplasmic reticulum (Cascio et al 2001;Stoltze et al 2000b). Therefore, the specificity of the immunoproteasome and the constitutive proteasome has not diverged at these positions (Fig.…”