Essential glycan-dependent interactions optimize MHC class I peptide loading (100.51)

Abstract

Abstract In this study we sought to better understand the role of the glycoprotein quality control machinery in the assembly of MHC (Major Histocompatibility Complex) class I molecules with high-affinity peptides. The lectin-like chaperone calreticulin (CRT) and the thiol oxidoreductase ERp57 participate in the final step of this process as part of the PLC (peptide-loading complex). To investigate the mechanism of peptide loading and roles of individual components, we reconstituted a PLC sub-complex, excluding TAP, from purified, recombinant proteins. The minimal components required for PLC assembly and function in vitro were ERp57 disulfide-linked to the class I-specific chaperone tapasin, CRT, and MHC class I complexes bearing monoglucosylated glycans. Mutations disrupting the interaction of CRT with either ERp57 or the class I glycan completely eliminated tapasin-mediated peptide loading. Using the purified system, we also provide direct evidence for a novel role of UGT1 (UDP-glucose: glycoprotein glucosyltransferase 1) in MHC class I assembly. Recombinant UGT1 preferentially recognized and re-glucosylated class I molecules associated with a suboptimal ligand and allowed PLC re-engagement and peptide exchange. Collectively, the data indicate that the interaction of MHC class I molecules with the PLC is regulated by its N-linked glycan, and that UGT1 contributes to the optimization of the MHC class I peptide repertoire in addition to tapasin.