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 endopla smic reticulum (ER) lumen, where they bind to nascent MHC class I mole cules. However, it is not known whether only high affinity peptides en ter the ER, or whether ER proteases must trim longer precursor peptide s down to optimal size. To evaluate the feasibility of proteolytic fin e trimming in vitro, soluble K-d and K-b were preincubated with peptid es that bind to K-d or K-b and the mixture was exposed to three differ ent proteases. Class I protected allele-specific peptides against prot eolysis, whereas the other peptides were degraded to the amino acid le vel. When a K-d/S11E (SYIPSAEYILE) complex was immunoprecipitated afte r incubation with carboxypeptidase, both S11E and the optimal sized S9 1 (SYIPSAEYI) were found to be specifically bound to K-d. However, onl y S91 was recovered if S11E, K-d and carboxypeptidase were mixed at th e same time, and there was no fine-trimming of bound S11E if high prot ease concentrations and short proteolysis times were used, which sugge sts that trimming occurs only in the unbound state. The amount of pept ide that immunoprecipitated with K-d increased after carboxypeptidase treatment of K-d/S11E, implying that the peptide affinity had increase d. K-d also protected S91 against proteolysis by a lysed microsome pre paration, demonstrating that class I could also protect high affinity peptides in vivo. These results suggest that class I participates in t he selection of high affinity peptides in the ER, by sampling transpor ted peptides for their affinity before unbound peptides are degraded b y ER proteases or efflux back to the cytosol.