A role for a novel luminal endoplasmic reticulum aminopeptidase in final trimming of 26 S proteasome-generated major histocompatability complex class I antigenic peptides.
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, ODC-LEova 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.
Komlosh
A
A
Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
Momburg
F
F
Weinschenk
T
T
Emmerich
N
N
Schild
H
H
Nadav
E
E
Shaked
I
I
Reiss
Y
Y
D000596:Amino Acids / Q000737:chemistry; D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D000818:Animals; D002460:Cell Line; D002474:Cell-Free System; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000201:enzymology* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000939:Epitopes; D005805:Genes, MHC Class I / Q000235:genetics*; D013058:Mass Spectrometry; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008861:Microsomes / Q000378:metabolism; D009955:Ornithine Decarboxylase / Q000737:chemistry* / Q000378:metabolism; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010618:Phenanthrolines / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors
D000596:Amino Acids; D004791:Enzyme Inhibitors; D000939:Epitopes; D010455:Peptides; D010618:Phenanthrolines; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D010447:Peptide Hydrolases; D000626:Aminopeptidases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D009955:Ornithine Decarboxylase; C025205:1,10-phenanthroline