A role for a novel luminal endoplasmic reticulum aminopeptidase in final trimming of 26 S proteasome-generated major histocompatability complex classI antigenic peptides

Peptides presented to cytotoxic, T lymphocytes by the class I major histoco mpatability complex are 8-11 residues long. Although proteasomal activity g enerates the precise G termini of antigenic epitopes, the mechanism(s) invo lved in generation of the precise N termini is largely unknown. To investig ate the mechanism of N-terminal. peptide processing, we used a cell-free sy stem in which two recombinant ornithine decarboxylase (ODC) constructs, one expressing the native H2-K-b-restricted ovalbumin (ova)-derived epitope SI INFEKL (ODC-ova) and the other expressing the extended epitope LESIINFEKL ( ODC-LEova), were targeted to degradation by 26 S proteasomes followed by im port into microsomes. We found that the cleavage specificity of the 26 S pr oteasome was influenced by the N-terminal flank ng amino acids leading to s ignificantly different yields of the final epitope SIINFEKL. Following incu bation in the presence of purified 26 S proteasome, ODC-LEova generated lar gely ESIINFEKL that was efficiently converted to the final epitope SIINFEKL following translocation into microsomes. The conversion of ESIINFEKL to SI INFEKL was strictly dependent on the presence of H2-K-b and was completely inhibited by the metalloaminopeptidase inhibitor 1,10-phenanthroline. Impor tantly, the converting activity was resistant, to a stringent salt/EDTA was h of the microsomes and was only apparent when transport of TAP, the transp orter associated with antigen processing, was facilitated. These results st rongly suggest a crucial role for a luminal endoplasmic reticulum-resident metalloaminopeptidase in the N-terminal trimming of major histocompatabilit y complex class I-associated peptides.