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

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-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).

Fig. 1. Subunit composition of the preparations of proteasomes and immunoproteasomes. 26S and 20S were analyzed by SDS–PAGE and western blot analysis with antibodies against different β-subunits.

Fig. 2. Ova degradation by proteasomes and immunoproteasomes. (A) Ova was incubated with SDS-activated 20S particles or with 26S particles in the presence of ATP. Aliquots were analyzed using fluorescamine. (B) 26S proteasomes or immunoproteasomes were incubated at 37°C for 9 h in the presence of Ova. The samples were then subjected to non-denaturing PAGE at 4°C, and the gel analyzed by overlay of fluorogenic substrate (100 µM Suc-LLVY-amc). The same gel was then Commassie Blue stained. Lanes 1 and 2, 26S proteasomes and immunoproteasomes. The two bands correspond to the double and single capped forms of the 26S particle. Lanes 4 and 7, 26S immunoproteasomes after 9 h of incubation at 37°C; lanes 5 and 8, 26S proteasomes after 9 h of incubation at 37°C; lanes 3 and 6, 20S as control.

Fig. 3. Sizes of peptides generated from Ova by 26S proteasomes and immunoproteasomes. After 9 h incubation, the peptide products were separated from the undegraded substrate on a C₁₈ column and then run on a polyhydroxyethyl aspartamide column. The molar amounts of peptides in each fraction were determined using fluorescamine. Each curve is an average of two independent incubations and peptide analyses.

Fig. 4. 26S proteasomes and immunoproteasomes generate different patterns of peptides from IGF-1. Denatured IGF-1 was incubated with 26S particles. Peptides generated were separated on a C₈ Vydac column equilibrated with 0.06% trifluoroacetic acid. Peptides were eluted at a flow rate of 0.15 ml/min with a gradient of acetonitrile from 0 to 8% in 20 min, to 28% in the next 100 min, to 36% in the subsequent 20 min, and to 44% in the last 10 min.

Fig. 5. 26S and 20S proteasomes and immunoproteasomes generate from Ova SIINFEKL and different amounts of N-extended variants. An aliquot of peptides generated by the proteasomes was loaded onto the antibody column, which binds peptides with the C-terminal sequence SIINFEKL. These eluted peptides were analyzed by HPLC.

Fig. 6. Immunoproteasomes generate similar amounts of SIINFEKL but greater amounts of N-extended versions than proteasomes. (A) All peptides analyzed increased at linear rates, as Ova was digested. At 3, 6 and 9 h, aliquots were analyzed for the content of SIINFEKL and N-extended versions. To measure the amounts of each peptide, the areas under individual peaks were compared with those obtained upon analyzing known amounts of each. (B) Yields of SIINFEKL and all N-extended versions combined. The data shown are the averages of three independent experiments for the 26S proteasomes and of two for the 20S particles. *SIINFEKL versus N-extended versions, p = 0.0495, Mann–Whitney U-test.