Discrete Cleavage Motifs of Constitutive and Immunoproteasomes Revealed by Quantitative Analysis of Cleavage Products

Abstract

Proteasomes are the main proteases responsible for cytosolic protein degradation and the production of major histocompatibility complex class I ligands. Incorporation of the interferon γ–inducible subunits low molecular weight protein (LMP)-2, LMP-7, and multicatalytic endopeptidase complex–like (MECL)-1 leads to the formation of immunoproteasomes which have been associated 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 peptide repertoire changing the overall cytotoxic T cell–specificity as indicated by the observation that LMP-7−/− mice react against cells of LMP-7 wild-type mice. Moreover, using the 436 amino acid protein enolase-1 as an unmodified model substrate in combination with a quantitative approach, we analyzed a large collection of peptides generated by either set of proteasomes. Inspection of the amino acids flanking proteasomal cleavage sites allowed the description of two different cleavage motifs. These motifs finally explain recent findings describing differential processing of epitopes by constitutive and immunoproteasomes and are important to the understanding of peripheral T cell tolerization/activation as well as for effective vaccine development.

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

“…To pinpoint functional differences between immunoproteasomes and constitutive proteasomes, we use the degradation data of Toes et al (2001). In short, proteasomes were purified from EBV-transformed B cells.…”

“…To calculate how often a possible site was actually used, the absolute amount of each peptide detected was determined by Edman sequencing. The details of the experimental procedure are described in Toes et al (2001). Toes et al (2001) calculated the frequency distributions of amino acids at cleavage sites and their flanking regions ( Table 2).…”

“…To predict cleavage sites in proteins we use amino acid frequencies occurring at and around the cleavage sites during degradation of yeast enolase by the immunoproteasome and the constitutive proteasome, as given in Toes et al (2001). In this paper we adapted the data for the immunoproteasome cleavages in Table 2.…”

“…We use the nomenclature suggested by Baumeister et al (1998); for some subunits the old nomenclature or original annotation is given in parenthesis. Abbreviations used in the phylogenetic trees are given after the organism names Toes et al (2001) for the C k values.These definitions are based on the assumption that each amino acid in the peptide contributes independently to the cleavage probability. We corrected the I k and C k values for the amino acid distribution in yeast enolase, and then ranked each position in 6,041 human and 1,304 pathogen proteins (compiled from Swissport version 39, see above) according to their P i and P c values.…”

“…Thus, the limited number of epitopes/proteins that are studied do not provide (1) a clear and quantitative picture of the specificity of the two proteasomes, (2) the effect of the specificity differences on a genome level (both for self and non-self) and (3) direct evidence that the immunoproteasome actually generates more antigenic peptides. Here we use a bioinformatics analysis to quantify the differences between the two forms of proteasomes based on the degradation of yeast enolase protein by human constitutive proteasome and immunoproteasome (Toes et al 2001). By using well-defined measures for diversity and information content, we are able to account for the small sample size and the biases that might rise from the choice of epitopes/proteins.…”