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

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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 efficiency 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‐γ‐induced β‐subunits and are more efficient 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 specificity of immunoproteasomes influence 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‐γ‐induced enzyme leucine aminopeptidase).