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. 2001 Jul 2;194(1):1-12.
doi: 10.1084/jem.194.1.1.

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

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Free PMC article

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

R E Toes et al. J Exp Med. .
Free PMC article

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 gamma--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.

Figures

Figure 1
Figure 1
Immunosubunit incorporation into 20S proteasomes purified from LCL-721 cells (left) but not into 20S proteasomes derived from LCL-721.174 cells (right). 20S proteasomes were isolated from LCL-721 and LCL-721.174 cells as described in Materials and Methods. Proteasome subunits were separated by SDS-PAGE (a) and probed with an LMP-7–specific antiserum (b). LMP-7 was only detected in 721 proteasomes. i or c indicate subunits exclusively present or overexpressed in i20S or c20S proteasomes, respectively.
Figure 1
Figure 1
Immunosubunit incorporation into 20S proteasomes purified from LCL-721 cells (left) but not into 20S proteasomes derived from LCL-721.174 cells (right). 20S proteasomes were isolated from LCL-721 and LCL-721.174 cells as described in Materials and Methods. Proteasome subunits were separated by SDS-PAGE (a) and probed with an LMP-7–specific antiserum (b). LMP-7 was only detected in 721 proteasomes. i or c indicate subunits exclusively present or overexpressed in i20S or c20S proteasomes, respectively.
Figure 2
Figure 2
Digestion map generated from degradation of enolase by constitutive proteasomes (a) and immunoproteasomes (b). Vertical lines, cleavage sites determined by Edman degradation and/or MS; black bars, degradation products identified by Edman degradation in combination with MS; white bars, degradation products identified by Edman degradation only (COOH terminus of peptide not identified).
Figure 2
Figure 2
Digestion map generated from degradation of enolase by constitutive proteasomes (a) and immunoproteasomes (b). Vertical lines, cleavage sites determined by Edman degradation and/or MS; black bars, degradation products identified by Edman degradation in combination with MS; white bars, degradation products identified by Edman degradation only (COOH terminus of peptide not identified).
Figure 3
Figure 3
Relative frequencies of amino acids in position P1 (a) P1′ (b). The absolute amount of amino acids found in a defined positions (P6 to P6′) around cleavage sites used by constitutive proteasomes and immuoproteasomes was divided by the total amount of peptides detected in the digests resulting in the relative frequency of amino acid usage in that position. Big white bars, relative frequency of amino acids found in enolase; black bars, relative frequency of amino acids at P1 or P1′ positions in peptide fragments generated by constitutive proteasomes; grey bars, relative frequency of amino acids at P1 or P1′ in peptide fragments generated by immunoproteasomes.
Figure 3
Figure 3
Relative frequencies of amino acids in position P1 (a) P1′ (b). The absolute amount of amino acids found in a defined positions (P6 to P6′) around cleavage sites used by constitutive proteasomes and immuoproteasomes was divided by the total amount of peptides detected in the digests resulting in the relative frequency of amino acid usage in that position. Big white bars, relative frequency of amino acids found in enolase; black bars, relative frequency of amino acids at P1 or P1′ positions in peptide fragments generated by constitutive proteasomes; grey bars, relative frequency of amino acids at P1 or P1′ in peptide fragments generated by immunoproteasomes.
Figure 5
Figure 5
Summary of cleavage motifs. Amino acid preferences at positions flanking the cleavages performed by constitutive (c20S) or immunoproteasomes (i20S) are indicated by bars pointing up (enriched) or down (decreased). Amino acid frequencies around cleavage sites were compared with amino acid frequencies in enolase. i20S versus c20S indicates a comparison of amino acid preferences between both types of proteasomes. Amino acid frequencies around cleavage sites were compared with each other. Bars pointing up indicate a i20S preference, bars pointing down indicate a c20S preference. Amino acids are shown in one-letter code by capital letters, and amino acid characteristics are indicated by small letters. A decrease in preferences for hydrophobic and bulky amino acids also indicates an increase in preferences for polar and small amino acids, respectively and vice versa. h, hydrophobic; f, flexible; b, bulky.
Figure 4
Figure 4
(a) Skin of the indicated donors were grafted on the back of the indicated recipient mice according to the protocol described in Materials and Methods. Graft rejection was controlled daily. Each dot represents a single recipient. This graph shows a representative result of four independent experiments. (b) LMP-7−/− mice and wild-type littermates (129/Ola background) were immunized with wild-type or LMP-7−/− splenocytes. CTL activity was tested after in vitro stimulation on LMP-7−/− and wild-type concanavalin A blasts, RMA, and RMA-S cells as described in Materials and Methods. This graph shows a representative result of three independent experiments.
Figure 4
Figure 4
(a) Skin of the indicated donors were grafted on the back of the indicated recipient mice according to the protocol described in Materials and Methods. Graft rejection was controlled daily. Each dot represents a single recipient. This graph shows a representative result of four independent experiments. (b) LMP-7−/− mice and wild-type littermates (129/Ola background) were immunized with wild-type or LMP-7−/− splenocytes. CTL activity was tested after in vitro stimulation on LMP-7−/− and wild-type concanavalin A blasts, RMA, and RMA-S cells as described in Materials and Methods. This graph shows a representative result of three independent experiments.

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