Discrete cleavage motifs of constitutive and immunoproteasomes revealed byquantitative analysis of cleavage products

Proteasomes are the main proteases responsible for cytosolic protein degrad ation and the production of major histocompatibility complex class I ligand s. Incorporation of the interferon gamma -inducible subunits low molecular weight protein (LMP)-2, LMP-7, and multicatalytic endopeptidase complex-lik e (MECL)-1 leads to the formation of immunoproteasomes which have been asso ciated with more efficient class I antigen processing. Although differences in cleavage specificities of constitutive and immunoproteasomes have been observed frequently, cleavage motifs have not been described previously. We now report that cells expressing immunoproteasomes display a different p eptide repertoire changing the overall cytotoxic T cell-specificity as indi cated by the observation that LMP-7(-/-) mice react again cells of LMP-7 wi ld type mice. Moreover, using the 436 amino acid protein enolase-1 as an un modified model substrate in combination with a quantitative approach, we an alyzed a large collection of peptides generated by either set of proteasome s. Inspection of the amino acids flanking proteasomal cleavage sites allowe d the description of two different cleavage motifs. These motifs finally ex plain recent findings describing differential processing of epitopes by con stitutive and immunoproteasomes and are important to the understanding of p eripheral T cell tolerization/activation as well as for effective vaccine d evelopment.