Role for MHC class I molecules in selecting and protecting high affinity peptides in the presence of proteases.
Ag fragments derived from the cytosol are transported into the endoplasmic reticulum (ER) lumen, where they bind to nascent MHC class I molecules. However, it is not known whether only high affinity peptides enter the ER, or whether ER proteases must trim longer precursor peptides down to optimal size. To evaluate the feasibility of proteolytic fine trimming in vitro, soluble Kd and Kb were preincubated with peptides that bind to Kd or Kb and the mixture was exposed to three different proteases. Class I protected allele-specific peptides against proteolysis, whereas the other peptides were degraded to the amino acid level. When a Kd/S11E (SYIPSAEYILE) complex was immunoprecipitated after incubation with carboxypeptidase, both S11E and the optimal sized S9I (SYIPSAEYI) were found to be specifically bound to Kd. However, only S91 was recovered if S11E, Kd and carboxypeptidase were mixed at the same time, and there was no fine-trimming of bound S11E if high protease concentrations and short proteolysis times were used, which suggests that trimming occurs only in the unbound state. The amount of peptide that immunoprecipitated with Kd increased after carboxypeptidase treatment of Kd/S11E, implying that the peptide affinity had increased. Kd also protected S9I against proteolysis by a lysed microsome preparation, demonstrating that class I could also protect high affinity peptides in vivo. These results suggest that class I participates in the selection of high affinity peptides in the ER, by sampling transported unbound peptides are degraded by ER proteases or efflux back to the cytosol.
Ojcius
D M
DM
Gene Molecular Biology Unit, Pasteur Institute, INSERM 277, Paris, France.
Langlade-Demoyen
P
P
Gachelin
G
G
Kourilsky
P
P
D000595:Amino Acid Sequence; D000818:Animals; D010450:Endopeptidases / Q000494:pharmacology*; D004721:Endoplasmic Reticulum / Q000201:enzymology; D006183:H-2 Antigens / Q000502:physiology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism*
D006183:H-2 Antigens; C024755:H-2K(K) antigen; D010455:Peptides; D010450:Endopeptidases