26S proteasomes and immunoproteasomes produce mainly N-extended versions of an antigenic peptide.
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-gamma-induced beta-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-gamma-induced enzyme leucine aminopeptidase).
Cascio
P
P
Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
Hilton
C
C
Kisselev
A F
AF
Rock
K L
KL
Goldberg
A L
AL
D000818:Animals; D000941:Antigens / Q000096:biosynthesis* / Q000276:immunology; D004527:Egg Proteins / Q000096:biosynthesis* / Q000276:immunology; D018985:Epitopes, B-Lymphocyte / Q000096:biosynthesis* / Q000276:immunology; D006825:Hybridomas; D016056:Immunodominant Epitopes / Q000096:biosynthesis* / Q000276:immunology; D051379:Mice; D010047:Ovalbumin / Q000096:biosynthesis* / Q000276:immunology; D010452:Peptide Biosynthesis; D010446:Peptide Fragments; D010447:Peptide Hydrolases / Q000378:metabolism*; D010455:Peptides / Q000276:immunology; D046988:Proteasome Endopeptidase Complex
D000941:Antigens; D004527:Egg Proteins; D018985:Epitopes, B-Lymphocyte; D016056:Immunodominant Epitopes; C110366:OVA-8; D010446:Peptide Fragments; D010455:Peptides; D010047:Ovalbumin; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease