Decrypting pMHCI Diversity Through Noncanonical Translation Initiation

Abstract

Abstract CD8 +T-cells rely on MHCI to surveil the proteomic state of cells in the form of peptide-MHCI (pMHCI). We previously uncovered a set of ATG-like translation initiation codons that can define noncanonical open reading frames (ORFs) of cryptic peptides presented by MHCI. The model cryptic codon CTG shares key translation initiation requirements with canonical ATG initiation, yet incorporates N-terminal Methionine (nMet) or Leucine (nLeu). We aim to characterize the activity and peptide products of the remaining cryptic codons to understand how cryptic ORFs contribute to the peptidome accessible to MHCI. Therefore, we make use of the subfemtomole sensitivity of the hyperB3Z reporter T-cell hybridoma to interrogate the identity of a modified OVA peptide (SIINFEHL;[SHL8]) reporter in a fixed cryptic initiation context, while retaining information on the residue incorporated during translation initiation. Following transfection of mammalian model cell lines with reporter, purified peptides from total cell extracts are RP-HPLC fractionated to differentiate N-terminally extended and trimmed [SHL8] intermediates. We found that nMet is incorporated by CTG and ACG initiation, while nLeu was absent under CTG initiation despite coexpression of the H2-D ballotype known to protect N-terminally extended [SHL8]. Peptide reporters with a P-2 residue derived from a codon satisfying the optimal (CACCxxx G) Kozak sequence (i.e. G, D) revealed that bulky P-2 residues enhance recovery of total reporter suggesting cryptic ORF peptide stability largely depends on P-2. Thus, by understanding the products of cryptic ORFs we expand the definition of immune-self while exposing new sources of pMHCI unique to autoimmunity and viral pathogens. Supported by grants from NIH (R01-AI130210, R01-AI121174, R37-AI060040)