A Role for a Novel Luminal Endoplasmic Reticulum Aminopeptidase in Final Trimming of 26 S Proteasome-generated Major Histocompatability Complex Class I Antigenic Peptides

Abstract

Peptides presented to cytotoxic T lymphocytes by the class I major histocompatability complex are 8 -11 residues long. Although proteasomal activity generates the precise C termini of antigenic epitopes, the mechanism(s) involved in generation of the precise N termini is largely unknown. To investigate the mechanism of N-terminal peptide processing, we used a cell-free system in which two recombinant ornithine decarboxylase (ODC) constructs, one expressing the native H2-K b -restricted ovalbumin (ova)-derived epitope SIINFEKL (ODC-ova) and the other expressing the extended epitope LESIINFEKL (ODC-LEova), were targeted to degradation by 26 S proteasomes followed by import into microsomes. We found that the cleavage specificity of the 26 S proteasome was influenced by the N-terminal flanking amino acids leading to significantly different yields of the final epitope SIINFEKL. Following incubation in the presence of purified 26 S proteasome, ODCLEova generated largely ESIINFEKL that was efficiently converted to the final epitope SIINFEKL following translocation into microsomes. The conversion of ESIINFEKL to SIINFEKL was strictly dependent on the presence of H2-K b and was completely inhibited by the metalloaminopeptidase inhibitor 1,10-phenanthroline. Importantly, the converting activity was resistant to a stringent salt/EDTA wash of the microsomes and was only apparent when transport of TAP, the transporter associated with antigen processing, was facilitated. These results strongly suggest a crucial role for a luminal endoplasmic reticulum-resident metalloaminopeptidase in the N-terminal trimming of major histocompatability complex class I-associated peptides. MHC1 class I molecules associate with peptides of 8 -11 amino acids derived from the proteolytic degradation of intracellular protein antigens and present them to CD8ϩ T cells on the cell surface (1-3). Although the vast majority of class I ligands are translocated from the cytosol into the endoplasmic reticulum (ER) by the ATP-dependent transporter associated with antigen processing (TAP) (3), alternative pathways have been described including the liberation of minigene-encoded peptides from ER signal sequences (4 -6), TAP-independent processing of signal sequences (7,8), and processing of membrane-associated or soluble proteins in the secretory pathway (9 -12). Peptides that are not retained in the ER by binding to class I or glycosylation are released back into the cytosol through the Sec61 channel (13-15). Studies using membrane-permeable inhibitors of proteasomes have indicated that the proteasome is the major proteolytic activity responsible for the generation of antigenic peptides (16 -21), although the incomplete inhibition of antigen processing by proteasome inhibitors has also demonstrated the involvement of nonproteasomal cytosolic proteases (22-28).The proteasome is an abundant cytosolic multisubunit protease consisting of the proteolytic 20 S core particle that associates with PA700 regulatory complexes to form 26 S proteasomes or with PA28 comp...

Leucine Aminopeptidase Is Not Essential for Trimming Peptides in the Cytosol or Generating Epitopes for MHC Class I Antigen Presentation

“…Proteasomes have been shown in vitro (3,35) and in vivo (10) to generate many N-extended precursors. Other experiments have shown that if such N-extended peptide precursors are expressed or injected into cells, they are trimmed and presented by MHC class I on the cell surface (11,14).…”

Cutting Edge: H-2Ld Class I Molecule Protects an HIV N-Extended Epitope from In Vitro Trimming by Endoplasmic Reticulum Aminopeptidase Associated with Antigen Processing

“…Previous studies showed that the enzymatic activity of ERAAP in microsomes or living cells trims peptide precursors in a short time period (minutes) (27)(28)(29). In contrast, in vitro ERAAP was more inefficient because longer incubations are required with the recombinant or purified enzyme (in the order of hours) (7).…”

The ER aminopeptidase, ERAP1, trims precursors to lengths of MHC class I peptides by a “molecular ruler” mechanism

“…TAP preferentially transports peptides 8-16 residues long (21) and seems more efficient in transporting Nextended precursors than the mature epitopes. A variety of studies have demonstrated that these precursors are trimmed by aminopeptidases in the ER lumen to the correct lengths for presentation on MHC class I molecules (22)(23)(24). Recently, an aminopeptidase, ERAP1 (ER aminopeptidase 1; ERAAP1), has been isolated by our lab (25) and Serwold et al (26) from the ER and shown to be responsible for trimming these N-extended precursors.…”