A role for a novel luminal endoplasmic reticulum aminopeptidase in final trimming of 26 S proteasome-generated major histocompatability complex classI antigenic peptides
A. Komlosh et al., A role for a novel luminal endoplasmic reticulum aminopeptidase in final trimming of 26 S proteasome-generated major histocompatability complex classI antigenic peptides, J BIOL CHEM, 276(32), 2001, pp. 30050-30056
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.